Anti-cd27 antibodies

ABSTRACT

The present invention relates to anti-CD27 antibodies, as well as use of these antibodies in the treatment of diseases such as cancer and infectious disease.

The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Sep. 20, 2017, is named 23413-US-NP_SL.txt and is 112,170 bytes in size.

FIELD OF THE INVENTION

The present invention relates to treatments of conditions ameliorated by stimulation of an immune response, in particular by the stimulation of antigen-specific T-lymphocytes. The various aspects of the present invention are suitable for treatment of any condition known or expected to be ameliorated by stimulation of CD27+ immune cells or by inhibition of one or more immune checkpoint protein(s). Conditions suitably treated by the invention are those ameliorated by immune stimulation, such as infectious diseases and cancers. More specifically the present invention relates to anti-CD27 antibodies, as well as use of these antibodies in the treatment of diseases such as cancer and infectious disease.

BACKGROUND OF THE INVENTION

CD27, a TNF receptor family member was identified as a membrane molecule on human T cells (van Lier et al., 1987, J. Immunol. 139:1589-96). According to current evidence, CD27 has a single ligand, CD70, which is also a TNF family member (Goodwin et al., 1993, Cell 73:447-56).

CD27 is exclusively expressed by hematopoietic cells, in particular those of the lymphocyte lineage, i.e. T-, B- and NK cells. CD27 was originally defined as a human T-cell co-stimulatory molecule that increments the proliferative response to TCR stimulation (van Lier et al., 1987, J. Immunol. 139:1589-96). Presence of CD70, the ligand of CD27, dictates the timing and persistence of CD27-mediated co-stimulation.

Transgenic expression of CD70 in immature dendritic cells sufficed to convert immunological tolerance to virus or tumors into CD8+ T cell responsiveness. Likewise, agonistic soluble CD70 promoted the CD8+ T cell response upon such peptide immunization (Rowley et al., 2004, J Immunol 172:6039-6046) and in CD70 transgenic mice, CD4+ and CD8+ effector cell formation in response to TCR stimulation was greatly facilitated (Arens et al. 2001, Immunity 15:801-12; Tesselaar et al., 2003, Nat Immunol 4:49-54; Keller et al. 2008, Immunity 29: 334-346). In mouse lymphoma models, tumor rejection was improved upon CD70 transgenesis or injection of an anti-mouse CD27 antibody (Arens et al., 2003, J Exp Med 199:1595-1605; French et al., 2007, Blood 109: 4810-15; Sakanishi and Yagita, 2010, Biochem. Biophys. Res. Comm. 393: 829-835; WO 2008/051424; WO 2012/004367).

In WO2012/004367 the first anti-human agonistic antibody (designated hCD27.15) was described that does not require crosslinking to activate CD27-mediated co-stimulation of the immune response. In addition, an anti-human CD27 antibody, designated 1F5 was disclosed that activates CD27 upon crosslinking (WO2011/130434 and Vitale et al., Clin. Cancer Res, 2012, 18(14): 3812-3821). However, there is still a need in the art to develop anti-human CD27 antibodies having improved characteristics, including the ability to bind human CD27 having the A59T SNP and CD27 from cynomolgus monkeys.

SUMMARY OF THE INVENTION

The invention provides anti-CD27 antibodies and antigen binding fragments thereof comprising the structural and functional features specified below.

In another embodiment, the invention provides an antibody or antigen binding fragment thereof that binds to human CD27 comprising a heavy chain variable region and a light chain variable region comprising: (i) a heavy chain variable region CDR1 comprising the amino acid sequence of SEQ ID NO: 1; (ii) a heavy chain variable region CDR2 comprising the amino acid sequence of SEQ ID NO: 2; and (iii) a heavy chain variable region CDR3 comprising the amino acid sequence of SEQ ID NO: 3. In one embodiment, the antibody or antigen binding fragment thereof comprises: (i) a heavy chain variable region CDR1 comprising the amino acid sequence of SEQ ID NO: 1, wherein X₁=M; (ii) a heavy chain variable region CDR2 comprising the amino acid sequence of SEQ ID NO: 2, wherein X₁=N, X₂=T, X₃=N and X₄=T; and (iii) a heavy chain variable region CDR3 comprising the amino acid sequence of SEQ ID NO: 3, wherein X₁=M. In one embodiment, the antibody or antigen binding fragment thereof optionally has at least one of the following characteristics: binds to human CD27 with an EC₅₀ of less than 100 pM, or less than 200 pM according to Cell ELISA assay, binds to human CD27 A59T with an EC₅₀ of less than 150 pM, or less than 250 pM according to Cell ELISA assay; binds to rhesus CD27 with an EC₅₀ of less than 100 pM, or less than 150 pM according to Cell ELISA assay; binds to human CD27 and human CD27 (A59T) with a bivalent KD value of about 5-10 nM as determined by surface plasmon resonance (e.g., BIACORE) or a similar technique (e.g. KinExa or OCTET); cross-reacts with cynomolgus or rhesus CD27; blocks binding of human CD27 to human CD70; increases T cell activation; stimulates antigen-specific T-cell production of IL-2 and IFNγ; induces NF-κB activation in human CD27 expressing cells with an EC50 of less than 5 nM when the antibody or fragment thereof is in soluble form; induces NF-κB activation in human CD27A59T expressing cells with an EC50 of less than 10 nM when the antibody or fragment thereof is in soluble form; induces NF-κB activation in Rhesus CD27 expressing cells with an EC50 of less than 1 nM when the antibody or fragment thereof is in soluble form; has an EC50 of less than 0.5 nM for binding to human CD27 on CD8+ or CD4+ T cells; increases CD8+ T cell activation in soluble form; and increases anti-CD3-induced IFN_(γ) production in human tumor culture. In one embodiment, the human CD27, human CD27A59T, or rhesus CD27 expressing cells are HEK293FT human embryonic kidney cells containing an NF-κB-luciferase reporter construct with CD27 plasmids transiently transfected. In one embodiment, the CD8+ T cell activation is measured by the % CD8+ T cells that are CD25+CD69+, and there is an average of about 1.5-2-fold increase in CD8+ T cell activation. In another embodiment, the increase in anti-CD3-induced IFN_(γ) production is at least 1.5 fold at 20 ug/ml of anti-CD27 antibody, and 10 ng/ml of anti-CD3 antibody.

In another embodiment, the invention provides an antibody or antigen binding fragment that binds to human CD27 comprising a heavy chain variable region and a light chain variable region comprising: (i) a light chain variable region CDR1 comprising the amino acid sequence of SEQ ID NO: 4, wherein X₁=M; (ii) a light chain variable region CDR2 comprising the amino acid sequence of SEQ ID NO: 5, wherein X₁=D and X₂=T; and (iii) a light chain variable region CDR3 comprising the amino acid sequence of SEQ ID NO: 6, wherein X₁=W, X₂=N and X₃=S. In one embodiment, the antibody or antigen binding fragment thereof optionally has at least one of the following characteristics: binds to human CD27 with an EC₅₀ of less than 100 pM, or less than 200 pM according to Cell ELISA assay, binds to human CD27 A59T with an EC₅₀ of less than 150 pM, or less than 250 pM according to Cell ELISA assay; binds to rhesus CD27 with an EC₅₀ of less than 100 pM, or less than 150 pM according to Cell ELISA assay; binds to human CD27 and human CD27 (A59T) with a bivalent KD value of about 5-10 nM as determined by surface plasmon resonance (e.g., BIACORE) or a similar technique (e.g. KinExa or OCTET); cross-reacts with cynomolgus or rhesus CD27; blocks binding of human CD27 to human CD70; increases T cell activation; stimulates antigen-specific T-cell production of IL-2 and IFNγ; induces NF-κB activation in human CD27 expressing cells with an EC50 of less than 5 nM when the antibody or fragment thereof is in soluble form; induces NF-κB activation in human CD27A59T expressing cells with an EC50 of less than 10 nM when the antibody or fragment thereof is in soluble form; induces NF-κB activation in Rhesus CD27 expressing cells with an EC50 of less than 1 nM when the antibody or fragment thereof is in soluble form; has an EC50 of less than 0.5 nM for binding to human CD27 on CD8+ or CD4+ T cells; increases CD8+ T cell activation in soluble form; and increases anti-CD3-induced IFN_(γ) production in human tumor culture. In one embodiment, the human CD27, human CD27A59T, or rhesus CD27 expressing cells are HEK293FT human embryonic kidney cells containing an NF-κB-luciferase reporter construct with CD27 plasmids transiently transfected. In one embodiment, the CD8+ T cell activation is measured by the % CD8+ T cells that are CD25+CD69+, and there is an average of about 1.5-2-fold increase in CD8+ T cell activation. In another embodiment, the increase in anti-CD3-induced IFN_(γ) production is at least 1.5 fold at 20 ug/ml of anti-CD27 antibody, and 10 ng/ml of anti-CD3 antibody.

In another embodiment of the invention, the antibody or antigen binding fragment comprises a heavy chain variable region and a light chain variable region comprising: a heavy chain variable region CDR1 comprising the amino acid sequence of SEQ ID NO: 1, wherein X₁=M; or an amino acid sequence differing from said sequence by 1, 2, or 3 conservative substitutions; a heavy chain variable region CDR2 comprising the amino acid sequence of SEQ ID NO: 2, wherein X₁=N, X₂=T, X₃=N and X₄=T, or an amino acid sequence differing from said sequence by 1, 2, or 3 conservative substitutions; a heavy chain variable region CDR3 comprising the amino acid sequence of SEQ ID NO: 3, wherein X₁=M, or an amino acid sequence differing from said sequence by 1, 2, or 3 conservative substitutions; a light chain variable region CDR1 comprising the amino acid sequence of SEQ ID NO: 4, wherein X₁=M, or an amino acid sequence differing from said sequence by 1, 2, or 3 conservative substitutions; a light chain variable region CDR2 comprising the amino acid sequence of SEQ ID NO: 5, wherein X₁=D and X₂=T, or an amino acid sequence differing from said sequence by 1, 2, or 3 conservative substitutions; and a light chain variable region CDR3 comprising the amino acid sequence of SEQ ID NO: 6, wherein X₁=W, X₂=N and X₃=S, or an amino acid sequence differing from said sequence by 1, 2, or 3 conservative substitutions. In one embodiment, the antibody or antigen binding fragment thereof optionally has at least one of the following characteristics: binds to human CD27 with an EC₅₀ of less than 100 pM, or less than 200 pM according to Cell ELISA assay, binds to human CD27 A59T with an EC₅₀ of less than 150 pM, or less than 250 pM according to Cell ELISA assay; binds to rhesus CD27 with an EC₅₀ of less than 100 pM, or less than 150 pM according to Cell ELISA assay; binds to human CD27 and human CD27 (A59T) with a bivalent KD value of about 5-10 nM as determined by surface plasmon resonance (e.g., BIACORE) or a similar technique (e.g. KinExa or OCTET); cross-reacts with cynomolgus or rhesus CD27; blocks binding of human CD27 to human CD70; increases T cell activation; stimulates antigen-specific T-cell production of IL-2 and IFNγ; induces NF-κB activation in human CD27 expressing cells with an EC50 of less than 5 nM when the antibody or fragment thereof is in soluble form; induces NF-κB activation in human CD27A59T expressing cells with an EC50 of less than 10 nM when the antibody or fragment thereof is in soluble form; induces NF-κB activation in Rhesus CD27 expressing cells with an EC50 of less than 1 nM when the antibody or fragment thereof is in soluble form; has an EC50 of less than 0.5 nM for binding to human CD27 on CD8+ or CD4+ T cells; increases CD8+ T cell activation in soluble form; and increases anti-CD3-induced IFN_(γ) production in human tumor culture. In one embodiment, the human CD27, human CD27A59T, or rhesus CD27 expressing cells are HEK293FT human embryonic kidney cells containing an NF-κB-luciferase reporter construct with CD27 plasmids transiently transfected. In one embodiment, the CD8+ T cell activation is measured by the % CD8+ T cells that are CD25+CD69+, and there is an average of about 1.5-2-fold increase in CD8+ T cell activation. In another embodiment, the increase in anti-CD3-induced IFN_(γ) production is at least 1.5 fold at 20 ug/ml of anti-CD27 antibody, and 10 ng/ml of anti-CD3 antibody.

In another embodiment of the invention, the antibody or antigen binding fragment comprises: an antibody or antigen binding fragment thereof that binds to human CD27 comprising a light chain immunoglobulin variable region, a heavy chain immunoglobulin variable region or both a light chain and a heavy chain immunoglobulin variable region selected from the group consisting of: an antibody or antigen binding fragment thereof comprising a variable heavy chain comprising the amino acid sequence of SEQ ID NO:7 or an amino acid sequence differing from SEQ ID NO: 7 by 1, 2, or 3 conservative substitutions and/or a variable light chain comprising the amino acid sequence of SEQ ID NO:8 or an amino acid sequence differing from SEQ ID NO: 8 by 1, 2, or 3 conservative substitutions; an antibody or antigen binding fragment thereof comprising a variable heavy chain comprising the amino acid sequence of SEQ ID NO:9 or an amino acid sequence differing from SEQ ID NO: 9 by 1, 2, or 3 conservative substitutions and/or a variable light chain comprising the amino acid sequence of SEQ ID NO: 14 or an amino acid sequence differing from SEQ ID NO: 14 by 1, 2, or 3 conservative substitutions; an antibody or antigen binding fragment thereof comprising a variable heavy chain comprising the amino acid sequence of SEQ ID NO:32 or an amino acid sequence differing from SEQ ID NO: 32 by 1, 2, or 3 conservative substitutions and/or a variable light chain comprising the amino acid sequence of SEQ ID NO:33 or an amino acid sequence differing from SEQ ID NO: 33 by 1, 2, or 3 conservative substitutions; an antibody or antigen binding fragment thereof comprising a variable heavy chain comprising the amino acid sequence of SEQ ID NO:34 or an amino acid sequence differing from SEQ ID NO: 34 by 1, 2, or 3 conservative substitutions and/or a variable light chain comprising the amino acid sequence of SEQ ID NO:35 or an amino acid sequence differing from SEQ ID NO: 35 by 1, 2, or 3 conservative substitutions; an antibody or antigen binding fragment thereof comprising a variable heavy chain comprising the amino acid sequence of SEQ ID NO:39 or an amino acid sequence differing from SEQ ID NO: 39 by 1, 2, or 3 conservative substitutions and/or a variable light chain comprising the amino acid sequence of SEQ ID NO:40 or an amino acid sequence differing from SEQ ID NO: 40 by 1, 2, or 3 conservative substitutions; an antibody or antigen binding fragment thereof comprising a variable heavy chain selected from the group consisting of SEQ ID NOs: 10-13 or an amino acid sequence differing from one of SEQ ID NOs: 10-13 by 1, 2, or 3 conservative substitutions and/or a variable light chain selected from the group consisting of any one of SEQ ID NOs: 15-18 or an amino acid sequence differing from one of SEQ ID NOs: 15-18 by 1, 2, or 3 conservative substitutions; an antibody or antigen binding fragment thereof comprising a variable heavy chain comprising the amino acid sequence of SEQ ID NO: 10 or an amino acid sequence differing from SEQ ID NO: 10 by 1, 2, or 3 conservative substitutions and/or a variable light chain comprising the amino acid sequence of SEQ ID NO: 15 or an amino acid sequence differing from SEQ ID NO: 15 by 1, 2, or 3 conservative substitutions. In one embodiment, the antibody or antigen binding fragment thereof optionally has at least one of the following characteristics: binds to human CD27 with an EC₅₀ of less than 100 pM, or less than 200 pM according to Cell ELISA assay, binds to human CD27 A59T with an EC₅₀ of less than 150 pM, or less than 250 pM according to Cell ELISA assay; binds to rhesus CD27 with an EC₅₀ of less than 100 pM, or less than 150 pM according to Cell ELISA assay; binds to human CD27 and human CD27 (A59T) with a bivalent KD value of about 5-10 nM as determined by surface plasmon resonance (e.g., BIACORE) or a similar technique (e.g. KinExa or OCTET); cross-reacts with cynomolgus or rhesus CD27; blocks binding of human CD27 to human CD70; increases T cell activation; stimulates antigen-specific T-cell production of IL-2 and IFNγ; induces NF-κB activation in human CD27 expressing cells with an EC50 of less than 5 nM when the antibody or fragment thereof is in soluble form; induces NF-κB activation in human CD27A59T expressing cells with an EC50 of less than 10 nM when the antibody or fragment thereof is in soluble form; induces NF-κB activation in Rhesus CD27 expressing cells with an EC50 of less than 1 nM when the antibody or fragment thereof is in soluble form; has an EC50 of less than 0.5 nM for binding to human CD27 on CD8+ or CD4+ T cells; increases CD8+ T cell activation in soluble form; and increases anti-CD3-induced IFN_(γ) production in human tumor culture. In one embodiment, the human CD27, human CD27A59T, or rhesus CD27 expressing cells are HEK293FT human embryonic kidney cells containing an NF-κB-luciferase reporter construct with CD27 plasmids transiently transfected. In one embodiment, the CD8+ T cell activation is measured by the % CD8+ T cells that are CD25+CD69+, and there is an average of about 1.5-2-fold increase in CD8+ T cell activation. In another embodiment, the increase in anti-CD3-induced IFN_(γ) production is at least 1.5 fold at 20 ug/ml of anti-CD27 antibody, and 10 ng/ml of anti-CD3 antibody.

In another embodiment, the invention provides an antibody or antigen binding fragment that binds to human CD27 comprising a heavy chain variable region and a light chain variable region comprising: (i) a heavy chain variable region CDR1 comprising the amino acid sequence of SEQ ID NO: 1; (ii) a heavy chain variable region CDR2 comprising the amino acid sequence of SEQ ID NO: 2; (iii) a heavy chain variable region CDR3 comprising the amino acid sequence of SEQ ID NO: 3; (iv) a light chain variable region CDR1 comprising the amino acid sequence of SEQ ID NO: 4; (v) a light chain variable region CDR2 comprising the amino acid sequence of SEQ ID NO: 5; and (vi) a light chain variable region CDR3 comprising the amino acid sequence of SEQ ID NO: 6. In another embodiment, the invention provides an antibody or antigen binding fragment that binds to human CD27 comprising a heavy chain variable region and a light chain variable region comprising: (i) a heavy chain variable region CDR1 comprising the amino acid sequence of SEQ ID NO: 1, wherein X₁=M; (ii) a heavy chain variable region CDR2 comprising the amino acid sequence of SEQ ID NO: 2, wherein X₁=N, X₂=T, X₃=N and X₄=T; (iii) a heavy chain variable region CDR3 comprising the amino acid sequence of SEQ ID NO: 3, wherein X₁=M; (iv) a light chain variable region CDR1 comprising the amino acid sequence of SEQ ID NO: 4, wherein X₁=M; (v) a light chain variable region CDR2 comprising the amino acid sequence of SEQ ID NO: 5, wherein X₁=D and X₂=T; and (vi) a light chain variable region CDR3 comprising the amino acid sequence of SEQ ID NO: 6, wherein X₁=W, X₂=N and X₃=S. In one embodiment, the antibody or antigen binding fragment thereof is humanized. In one embodiment, the antibody or antigen binding fragment thereof comprises a heavy chain variable region selected from the group consisting of SEQ ID NOs: 9, 10, 11, 12, 13, 32, 34 and 39; and a light chain variable region selected from the group consisting of SEQ ID NOs: 14, 15, 16, 17, 18, 33, 35 and 40. In one embodiment, the antibody or antigen binding fragment thereof comprises a heavy chain variable region comprising at least 90%, 95%, 96%, 97%, 98% or 99% identity to any one of SEQ ID NOs: 10, 11, 12 or 13; and a light chain variable region comprising at least 90%, 95%, 96%, 97%, 98% or 99% identity to any one of SEQ ID NOs: 15, 16, 17 or 18. In one embodiment, the antibody or antigen binding fragment thereof comprises a heavy chain variable region comprising 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions with respect to any one of SEQ ID NOs: 10, 11, 12 or 13; and a light chain variable region comprising 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions with respect to any one of SEQ ID NOs: 15, 16, 17 or 18. In one embodiment, the antibody or antigen binding fragment thereof optionally has at least one of the following characteristics: binds to human CD27 with an EC₅₀ of less than 100 pM, or less than 200 pM according to Cell ELISA assay, binds to human CD27 A59T with an EC₅₀ of less than 150 pM, or less than 250 pM according to Cell ELISA assay; binds to rhesus CD27 with an EC₅₀ of less than 100 pM, or less than 150 pM according to Cell ELISA assay; binds to human CD27 and human CD27 (A59T) with a bivalent KD value of about 5-10 nM as determined by surface plasmon resonance (e.g., BIACORE) or a similar technique (e.g. KinExa or OCTET); cross-reacts with cynomolgus or rhesus CD27; blocks binding of human CD27 to human CD70; increases T cell activation; stimulates antigen-specific T-cell production of IL-2 and IFNγ; induces NF-κB activation in human CD27 expressing cells with an EC50 of less than 5 nM when the antibody or fragment thereof is in soluble form; induces NF-κB activation in human CD27A59T expressing cells with an EC50 of less than 10 nM when the antibody or fragment thereof is in soluble form; induces NF-κB activation in Rhesus CD27 expressing cells with an EC50 of less than 1 nM when the antibody or fragment thereof is in soluble form; has an EC50 of less than 0.5 nM for binding to human CD27 on CD8+ or CD4+ T cells; increases CD8+ T cell activation in soluble form; and increases anti-CD3-induced IFN, production in human tumor culture. In one embodiment, the human CD27, human CD27A59T, or rhesus CD27 expressing cells are HEK293FT human embryonic kidney cells containing an NF-κB-luciferase reporter construct with CD27 plasmids transiently transfected. In one embodiment, the CD8+ T cell activation is measured by the % CD8+ T cells that are CD25+CD69+, and there is an average of about 1.5-2-fold increase in CD8+ T cell activation. In another embodiment, the increase in anti-CD3-induced IFN_(γ) production is at least 1.5 fold at 20 ug/ml of anti-CD27 antibody, and 10 ng/ml of anti-CD3 antibody.

In another embodiment, the invention provides an antibody or antigen binding fragment that binds to human CD27 comprising at least 90%, 95%, 96%, 97%, 98% or 99% identity to any one of SEQ ID NOs: 10, 11, 12 or 13; and a light chain variable region comprising at least 90%, 95%, 96%, 97%, 98% or 99% identity to any one of SEQ ID NOs: 15, 16, 17 or 18. In one embodiment, the proposed sequence variations occur only in the framework regions of the antibody.

In another embodiment, the invention provides an antibody or antigen binding fragment that binds to human CD27 comprising a heavy chain variable region and a light chain variable region comprising: (i) a heavy chain variable region CDR1 comprising the amino acid sequence of SEQ ID NO: 1; (ii) a heavy chain variable region CDR2 comprising the amino acid sequence of SEQ ID NO: 2; (iii) a heavy chain variable region CDR3 comprising the amino acid sequence of SEQ ID NO: 3; (iv) a light chain variable region CDR1 comprising the amino acid sequence of SEQ ID NO: 4; (v) a light chain variable region CDR2 comprising the amino acid sequence of SEQ ID NO: 5; and (vi) a light chain variable region CDR3 comprising the amino acid sequence of SEQ ID NO: 6; wherein the antibody or antigen binding fragment thereof comprises a heavy chain variable region comprising at least 90%, 95%, 96%, 97%, 98% or 99% identity to a heavy chain variable region selected from the group consisting of SEQ ID NOs: 9-13 and a light chain variable region comprising at least 90%, 95%, 96%, 97%, 98% or 99% identity to a light chain variable region selected from the group consisting of SEQ ID NOs: 14-18. In this aforementioned embodiment, the sequence variations occur in the framework regions, and the antibody or antigen binding fragment thereof comprises: (i) a heavy chain variable region CDR1 comprising the amino acid sequence of SEQ ID NO: 1, wherein X₁=M; (ii) a heavy chain variable region CDR2 comprising the amino acid sequence of SEQ ID NO: 2, wherein X₁=N, X₂=T, X₃=N and X₄=T; (iii) a heavy chain variable region CDR3 comprising the amino acid sequence of SEQ ID NO: 3, wherein X₁=M; (iv) a light chain variable region CDR1 comprising the amino acid sequence of SEQ ID NO: 4, wherein X₁=M; (v) a light chain variable region CDR2 comprising the amino acid sequence of SEQ ID NO: 5, wherein X₁=D and X₂=T; and (vi) a light chain variable region CDR3 comprising the amino acid sequence of SEQ ID NO: 6, wherein X₁=W, X₂=N and X₃=S. In one embodiment, the antibody or antigen binding fragment thereof optionally has at least one of the following characteristics: binds to human CD27 with an EC₅₀ of less than 100 pM, or less than 200 pM according to Cell ELISA assay, binds to human CD27 A59T with an EC₅₀ of less than 150 pM, or less than 250 pM according to Cell ELISA assay; binds to rhesus CD27 with an EC₅₀ of less than 100 pM, or less than 150 pM according to Cell ELISA assay; binds to human CD27 and human CD27 (A59T) with a bivalent KD value of about 5-10 nM as determined by surface plasmon resonance (e.g., BIACORE) or a similar technique (e.g. KinExa or OCTET); cross-reacts with cynomolgus or rhesus CD27; blocks binding of human CD27 to human CD70; increases T cell activation; stimulates antigen-specific T-cell production of IL-2 and IFNγ; induces NF-κB activation in human CD27 expressing cells with an EC50 of less than 5 nM when the antibody or fragment thereof is in soluble form; induces NF-κB activation in human CD27A59T expressing cells with an EC50 of less than 10 nM when the antibody or fragment thereof is in soluble form; induces NF-κB activation in Rhesus CD27 expressing cells with an EC50 of less than 1 nM when the antibody or fragment thereof is in soluble form; has an EC50 of less than 0.5 nM for binding to human CD27 on CD8+ or CD4+ T cells; increases CD8+ T cell activation in soluble form; and increases anti-CD3-induced IFN, production in human tumor culture. In one embodiment, the human CD27, human CD27A59T, or rhesus CD27 expressing cells are HEK293FT human embryonic kidney cells containing an NF-κB-luciferase reporter construct with CD27 plasmids transiently transfected. In one embodiment, the CD8+ T cell activation is measured by the % CD8+ T cells that are CD25+CD69+, and there is an average of about 1.5-2-fold increase in CD8+ T cell activation. In another embodiment, the increase in anti-CD3-induced IFN_(γ) production is at least 1.5 fold at 20 ug/ml of anti-CD27 antibody, and 10 ng/ml of anti-CD3 antibody.

In yet a further embodiment of the invention, it is provided an antibody or antigen binding fragment thereof that binds to human CD27 comprising both a light chain and a heavy chain immunoglobulin variable region selected from the group consisting of: an antibody or antigen binding fragment thereof comprising a variable heavy chain comprising the amino acid sequence of SEQ ID NO:7 and a variable light chain comprising the amino acid sequence of SEQ ID NO:8; an antibody or antigen binding fragment thereof comprising a variable heavy chain comprising the amino acid sequence of SEQ ID NO:9 and a variable light chain comprising the amino acid sequence of SEQ ID NO: 14; an antibody or antigen binding fragment thereof comprising a variable heavy chain comprising the amino acid sequence of SEQ ID NO:32 and a variable light chain comprising the amino acid sequence of SEQ ID NO:33; an antibody or antigen binding fragment thereof comprising a variable heavy chain comprising the amino acid sequence of SEQ ID NO:34 and a variable light chain comprising the amino acid sequence of SEQ ID NO:35; an antibody or antigen binding fragment thereof comprising a variable heavy chain comprising the amino acid sequence of SEQ ID NO:39 and a variable light chain comprising the amino acid sequence of SEQ ID NO:40; an antibody or antigen binding fragment thereof comprising a variable heavy chain selected from the group consisting of SEQ ID NOs: 10-13 and a variable light chain selected from the group consisting of SEQ ID NOs: 15-18; an antibody or antigen binding fragment thereof comprising a variable heavy chain comprising the amino acid sequence of SEQ ID NO: 10 and a variable light chain comprising the amino acid sequence of SEQ ID NO: 15; an antibody or antigen binding fragment that comprises the variable heavy chain of any one of SEQ ID NOs:10-12 and the variable light chain of any one of SEQ ID NOs:15-17; and an antibody or antigen binding fragment that comprises the variable heavy chain of SEQ ID NO:13 and the variable light chain of SEQ ID NO:18.

In one embodiment, the antibody or antigen binding fragment thereof optionally has at least one of the following characteristics: binds to human CD27 with an EC₅₀ of less than 100 pM, or less than 200 pM according to Cell ELISA assay, binds to human CD27 A59T with an EC₅₀ of less than 150 pM, or less than 250 pM according to Cell ELISA assay; binds to rhesus CD27 with an EC₅₀ of less than 100 pM, or less than 150 pM according to Cell ELISA assay; binds to human CD27 and human CD27 (A59T) with a bivalent KD value of about 5-10 nM as determined by surface plasmon resonance (e.g., BIACORE) or a similar technique (e.g. KinExa or OCTET); cross-reacts with cynomolgus or rhesus CD27; blocks binding of human CD27 to human CD70; increases T cell activation; stimulates antigen-specific T-cell production of IL-2 and IFNγ; induces NF-κB activation in human CD27 expressing cells with an EC50 of less than 5 nM when the antibody or fragment thereof is in soluble form; induces NF-κB activation in human CD27A59T expressing cells with an EC50 of less than 10 nM when the antibody or fragment thereof is in soluble form; induces NF-κB activation in Rhesus CD27 expressing cells with an EC50 of less than 1 nM when the antibody or fragment thereof is in soluble form; has an EC50 of less than 0.5 nM for binding to human CD27 on CD8+ or CD4+ T cells; increases CD8+ T cell activation in soluble form; and increases anti-CD3-induced IFN_(γ) production in human tumor culture. In one embodiment, the human CD27, human CD27A59T, or rhesus CD27 expressing cells are HEK293FT human embryonic kidney cells containing an NF-κB-luciferase reporter construct with CD27 plasmids transiently transfected. In one embodiment, the CD8+ T cell activation is measured by the % CD8+ T cells that are CD25+CD69+, and there is an average of about 1.5-2-fold increase in CD8+ T cell activation. In another embodiment, the increase in anti-CD3-induced IFN_(γ) production is at least 1.5 fold at 20 ug/ml of anti-CD27 antibody, and 10 ng/ml of anti-CD3 antibody.

In another embodiment, the invention provides an antibody or antigen binding fragment thereof, comprising: a variable heavy chain comprising the amino acid sequence of SEQ ID NO: 7 and/or a variable light chain comprising the amino acid sequence of SEQ ID NO: 8, wherein the antibody or antigen binding fragment thereof binds to human CD27. In one embodiment, the invention relates to an isolated antibody or antigen binding fragment that binds to human CD27 comprising: a heavy chain comprising the amino acid sequence of SEQ ID NO: 7 or variant thereof comprising up to 25 amino acid substitutions, and/or a light chain comprising the amino acid sequence of SEQ ID NO: 8 comprising up to 25 amino acid substitutions. In one embodiment, the antibody or antigen binding fragment thereof optionally has at least one of the following characteristics: binds to human CD27 with an EC₅₀ of less than 100 pM, or less than 200 pM according to Cell ELISA assay, binds to human CD27 A59T with an EC₅₀ of less than 150 pM, or less than 250 pM according to Cell ELISA assay; binds to rhesus CD27 with an EC₅₀ of less than 100 pM, or less than 150 pM according to Cell ELISA assay; binds to human CD27 and human CD27 (A59T) with a bivalent KD value of about 5-10 nM as determined by surface plasmon resonance (e.g., BIACORE) or a similar technique (e.g. KinExa or OCTET); cross-reacts with cynomolgus or rhesus CD27; blocks binding of human CD27 to human CD70; increases T cell activation; stimulates antigen-specific T-cell production of IL-2 and IFNγ; induces NF-κB activation in human CD27 expressing cells with an EC50 of less than 5 nM when the antibody or fragment thereof is in soluble form; induces NF-κB activation in human CD27A59T expressing cells with an EC50 of less than 10 nM when the antibody or fragment thereof is in soluble form; induces NF-κB activation in Rhesus CD27 expressing cells with an EC50 of less than 1 nM when the antibody or fragment thereof is in soluble form; has an EC50 of less than 0.5 nM for binding to human CD27 on CD8+ or CD4+ T cells; increases CD8+ T cell activation in soluble form; and increases anti-CD3-induced IFN_(γ) production in human tumor culture. In one embodiment, the human CD27, human CD27A59T, or rhesus CD27 expressing cells are HEK293FT human embryonic kidney cells containing an NF-κB-luciferase reporter construct with CD27 plasmids transiently transfected. In one embodiment, the CD8+ T cell activation is measured by the % CD8+ T cells that are CD25+CD69+, and there is an average of about 1.5-2-fold increase in CD8+ T cell activation. In another embodiment, the increase in anti-CD3-induced IFN_(γ) production is at least 1.5 fold at 20 ug/ml of anti-CD27 antibody, and 10 ng/ml of anti-CD3 antibody.

In any of the above embodiments, the antibody or antigen binding fragment thereof can be isolated.

In any of the above embodiments, the antibody or antigen binding fragment thereof is a recombinant antibody.

In any of the above embodiments, the antibody can be a full-length antibody.

In any of the above embodiments, the antibody or antigen binding fragment thereof can be a humanized antibody.

In any of the above embodiments, the antibody or antigen binding fragment thereof can be a humanized antibody comprising two heavy chains and two light chains, and optionally is an intact IgG antibody. In one embodiment, the heavy chains are of the IgG isotype. In one embodiment, the heavy chains are of the IgG1 isotype. In another embodiment, the heavy chains are of the IgG2 isotype. In another embodiment, the heavy chains are of the IgG4 isotype. In another embodiment, the heavy chains are of the IgM isotype. In one embodiment, the antibody comprises the heavy chain constant domain of SEQ ID NO: 30 or SEQ ID NO: 28.

In another aspect of the invention, the antibody or antigen binding fragment thereof comprises a heavy chain variable region and a light chain variable region comprising: (i) a heavy chain variable region CDR1 comprising the amino acid sequence of SEQ ID NO: 1, wherein X₁=M; (ii) a heavy chain variable region CDR2 comprising the amino acid sequence of SEQ ID NO: 2, wherein X₁=N, X₂=T, X₃=N and X₄=T; (iii) a heavy chain variable region CDR3 comprising the amino acid sequence of SEQ ID NO: 3, wherein X₁=M; (iv) a light chain variable region CDR1 comprising the amino acid sequence of SEQ ID NO: 4, wherein X₁=M; (v) a light chain variable region CDR2 comprising the amino acid sequence of SEQ ID NO: 5, wherein X₁=D and X₂=T; and (vi) a light chain variable region CDR3 comprising the amino acid sequence of SEQ ID NO: 6, wherein X₁=W, X₂=N and X₃=S, and the heavy chain constant domain is IgG1 or IgG4 isotype. In one embodiment, the antibody or antigen binding fragment thereof optionally has at least one of the following characteristics: binds to human CD27 with an EC₅₀ of less than 100 pM, or less than 200 pM according to cell ELISA assay, binds to human CD27 A59T with an EC₅₀ of less than 150 pM, or less than 250 pM according to Cell ELISA assay; binds to rhesus CD27 with an EC₅₀ of less than 100 pM, or less than 150 pM according to cell ELISA assay; binds to human CD27 and human CD27 (A59T) with a bivalent KD value of about 5-10 nM as determined by surface plasmon resonance (e.g., BIACORE) or a similar technique (e.g. KinExa or OCTET); cross-reacts with cynomolgus monkey or rhesus monkey CD27; blocks binding of human CD27 to human CD70; increases T cell activation; stimulates antigen-specific T-cell production of IL-2 and IFNγ; induces NF-κB activation in human CD27-expressing cells with an EC50 of less than 5 nM when the antibody or fragment thereof is in soluble form; induces NF-κB activation in human CD27A59T-expressing cells with an EC50 of less than 10 nM when the antibody or fragment thereof is in soluble form; induces NF-κB activation in rhesus monkey CD27 expressing cells with an EC50 of less than 1 nM when the antibody or fragment thereof is in soluble form; has an EC50 of less than 0.5 nM for binding to human CD27 on CD8+ or CD4+ T cells; increases CD8+ T cell activation in soluble form; and increases anti-CD3-induced IFN_(γ) production in human tumor culture. In one embodiment, the human CD27, human CD27A59T, or rhesus CD27 expressing cells are HEK293FT human embryonic kidney cells containing an NF-κB-luciferase reporter construct with CD27 plasmids transiently transfected. In one embodiment, the CD8+ T cell activation is measured by the % CD8+ T cells that are CD25+CD69+, and there is an average of about 1.5-2-fold increase in CD8+ T cell activation. In another embodiment, the increase in anti-CD3-induced IFN, production is at least 1.5 fold at 20 ug/ml of anti-CD27 antibody, and 10 ng/ml of anti-CD3 antibody.

In any of the above mentioned embodiments, the antibody or antigen binding fragment thereof of the invention can comprise a heavy chain region consisting of: (a) any of the variable heavy chains described above and (b) a leader peptide (for example, the leader peptide of SEQ ID NO: 26). In any of the above mentioned embodiments, the antibody or antigen binding fragment thereof of the invention can comprise a light chain region consisting of: (a) any of the light chains described above and (b) a leader peptide (for example, the leader peptide of SEQ ID NO: 27).

In any of the above mentioned embodiments of the invention, the antibody or antigen binding fragment thereof can be an antibody comprising any of the heavy chain variable regions described above and any human heavy chain constant domain. In one embodiment, the antibody or antigen binding fragment thereof is of the IgG isotype, and comprises a human IgG1, IgG2, IgG3 or IgG4 human heavy chain constant domain. In one embodiment, the antibody or antigen binding fragment thereof of the invention comprises a human heavy chain IgG1 constant domain (SEQ ID NO: 30) or a variant thereof, wherein the variant comprises up to 20 amino acid substitutions relative to SEQ ID NO:30. In another embodiment, the antibody or antigen binding fragment thereof comprises a human heavy chain IgG4 constant domain, wherein the amino acid at position 228 (using EU numbering scheme) has been substituted from Ser to Pro (SEQ ID NO: 28). In another embodiment, the heavy chains are of the IgM isotype.

In any of the above mentioned embodiments, the antibody or antigen binding fragment thereof can comprise any of the light chain variable regions described above and a human light chain constant domain. In one embodiment, the antibody or antigen binding fragment thereof comprises a human kappa light chain constant domain comprising the amino acid sequence of SEQ ID NO: 29.

In one embodiment, the anti-CD27 antibody of the invention comprises a full tetrameric structure having two light chains and two heavy chains, wherein each light chain comprises: a variable region comprising any one of SEQ ID NOs: 14-18, 33, 35 and 40 and a human kappa light chain or a human lambda light chain constant region; and each heavy chain comprises: a variable region comprising any one of SEQ ID NOs: 9-13, 32, 34 and 39, and a human IgG1 constant region (SEQ ID NO: 30).

In one embodiment, the anti-CD27 antibody of the invention comprises a full tetrameric structure having two light chains and two heavy chains, wherein each light chain comprises: a variable region comprising any one of SEQ ID NOs: 14-18, 33, 35 and 40 and a human kappa light chain or a human lambda light chain constant domain; and each heavy chain comprises: a variable region comprising any one of SEQ ID NOs: 9-13, 32, 34 and 39, and a human IgM constant region.

In one embodiment, the anti-CD27 antibody of the invention comprises a full tetrameric structure having two light chains and two heavy chains, wherein each light chain comprises: a variable region comprising any one of SEQ ID NOs: 14-18, 33, 35 and 40 and a human kappa light chain or a human lambda light chain constant domain; and each heavy chain comprises: a variable region comprising any one of SEQ ID NOs: 9-13, 32, 34 and 39, and a human IgG2 constant region.

In one embodiment, the anti-CD27 antibody of the invention comprises two light chains and two heavy chains, wherein each light chain consists of SEQ ID NO: 36; and each heavy chain consists of SEQ ID NO: 37.

In one embodiment, the anti-CD27 antibody of the invention consists of two light chains and two heavy chains, wherein each light chain consists of SEQ ID NO: 36; and each heavy chain consists of SEQ ID NO: 37.

In one embodiment, the anti-CD27 antibody of the invention comprises two light chains and two heavy chains, wherein each light chain consists of SEQ ID NO: 36; and each heavy chain consists of SEQ ID NO: 38.

In one embodiment, the anti-CD27 antibody of the invention consists of two light chains and two heavy chains, wherein each light chain consists of SEQ ID NO: 36; and each heavy chain consists of SEQ ID NO: 38.

In one embodiment, the anti-CD27 antibody of the invention comprises two light chains and two heavy chains, wherein each light chain consists of: a variable region comprising any one of SEQ ID NOs: 14-18, 33, 35 and 40 and a human kappa light chain or a human lambda light chain constant domain; and each heavy chain consists of: a variable region consisting of any one of SEQ ID NOs: 9-13, 32, 34 and 39 and a human IgM constant region.

In one embodiment, the anti-CD27 antibody of the invention consists of two light chains and two heavy chains, wherein each light chain consists of: a variable region comprising any one of SEQ ID NOs: 14-18, 33, 35 and 40 and a human kappa light chain or a human lambda light chain constant domain; and each heavy chain consists of: a variable region consisting of any one of SEQ ID NOs: 9-13, 32, 34 and 39 and a human IgM constant region.

In one embodiment, the anti-CD27 antibody of the invention comprises two light chains and two heavy chains, wherein each light chain consists of: a variable region consisting of any one of SEQ ID NOs: 14-18, 33, 35 and 40 and a human kappa light chain or a human lambda light chain constant domain; and each heavy chain consists of: a variable region comprising any one of SEQ ID NOs: 9-13, 32, 34 and 39, and a human IgG1 constant region.

In one embodiment, the anti-CD27 antibody of the invention consists of two light chains and two heavy chains, wherein each light chain consists of: a variable region consisting of any one of SEQ ID NOs: 14-18, 33, 35 and 40 and a human kappa light chain or a human lambda light chain constant domain; and each heavy chain consists of: a variable region comprising any one of SEQ ID NOs: 9-13, 32, 34 and 39, and a human IgG1 constant region.

In another aspect of the invention, any of the above antibody or antigen binding fragments comprises a glycosylation pattern characteristic of expression by a mammalian cell or CHO cell.

In certain embodiments, the anti-CD27 antibody or antigen binding fragment thereof is conjugated to at least one therapeutic agent. In one embodiment, the therapeutic agent is a second antibody or fragment thereof, an immunomodulator, a hormone, a cytotoxic agent, an enzyme, a radionuclide, a second antibody conjugated to at least one immunomodulator, enzyme, radioactive label, hormone, antisense oligonucleotide, or cytotoxic agent, or a combination thereof.

The invention also provides isolated polypeptides comprising the amino acid sequence of any one of SEQ ID NOs: 1-18, 32-40 and 44-45 or a fragment of any of said sequences.

The invention also provides isolated nucleic acids encoding anyone of the anti-CD27 antibodies or antigen binding fragments of the invention. In one embodiment, the invention provides isolated nucleic acids encoding any one of the polypeptides of SEQ ID NOs: 1-18, 32-40 and 44-45, wherein said polypeptides can optionally comprise a leader sequence. In another embodiment, the invention provides an isolated nucleic acid comprising SEQ ID NO: 46 or SEQ ID NO: 47, or both. The invention also provides expression vectors comprising a nucleic acid encoding any one of the polypeptides of SEQ ID NOs: 1-18, 32-40 and 44-45 (wherein said polypeptides can optionally comprise a leader sequence) or a nucleic acid comprising SEQ ID NO: 46 or SEQ ID NO: 47, or both. These isolated nucleic acids and the expression vectors comprising them may be used to express the antibodies of the invention or antigen binding fragments thereof in recombinant host cells. Thus, the invention also provides host cells comprising nucleic acids encoding any one of the polypeptides of SEQ ID NOs: 1-18, 32-40 and 44-45 (wherein said polypeptides can optionally comprise a leader sequence) or a nucleic acid comprising SEQ ID NO: 46 or SEQ ID NO: 47, or both. In one embodiment, the host cell is a bacterial cell, a human cell, a mammalian cell, a Pichia cell, a plant cell, an HEK293 cell, or a Chinese hamster ovary cell. In one embodiment, the host cell is Chinese hamster ovary cell. In one embodiment, the host cell is a yeast cell, for example a Pichia cell or a Pichia pastoris host cell.

The invention also provides pharmaceutical compositions comprising an antibody or antigen binding fragment of the invention and a pharmaceutically acceptable carrier or diluent. In one embodiment, the composition comprises a further therapeutic agent. In one embodiment, the further therapeutic agent is selected from the group consisting of: an anti-LAG3 antibody or an antigen binding fragment thereof; an anti-TIGIT antibody or antigen biding fragment thereof; an anti-VISTA antibody or an antigen binding fragment thereof; an anti-BTLA antibody or an antigen binding fragment thereof; an anti-TIM3 antibody or an antigen binding fragment thereof; an anti-CTLA4 antibody or an antigen binding fragment thereof; an anti-HVEM antibody or an antigen binding fragment thereof; an anti-CD70 antibody or an antigen binding fragment thereof; an anti-OX40 antibody or an antigen binding fragment thereof; an anti-CD28 antibody or an antigen binding fragment thereof; an anti-PD1 antibody or an antigen binding fragment thereof; an anti-PDL1 antibody or an antigen binding fragment thereof; an anti-PDL2 antibody or an antigen binding fragment thereof; an anti-GITR antibody or an antigen binding fragment thereof; an anti-ICOS antibody or an antigen binding fragment thereof; an anti-SIRPα antibody or an antigen binding fragment thereof; an anti-ILT2 antibody or antigen binding fragment thereof; an anti-ILT3 antibody or antigen binding fragment thereof; an anti-ILT4 antibody or antigen binding fragment thereof; and an anti-ILT5 antibody or an antigen binding fragment thereof; an anti 4-1BB (CD137) antibody or an antigen binding fragment thereof; an anti-NKG2A antibody or an antigen binding fragment thereof; an anti-NKG2C antibody or an antigen binding fragment thereof; an anti-NKG2E antibody or an antigen binding fragment thereof; an anti-TSLP antibody or an antigen binding fragment thereof; an anti-IL-10 antibody or an antigen binding fragment thereof; IL-10 or PEGylated IL-10; a STING agonist; a CXCR2 antagonist; and a PARP inhibitor.

In one embodiment of the pharmaceutical compositions of the invention, the anti-CD27 antibody or antigen binding fragment of the invention comprises a heavy chain variable region and a light chain variable region comprising: (i) a heavy chain variable region CDR1 comprising the amino acid sequence of SEQ ID NO: 1; (ii) a heavy chain variable region CDR2 comprising the amino acid sequence of SEQ ID NO: 2; (iii) a heavy chain variable region CDR3 comprising the amino acid sequence of SEQ ID NO: 3; (iv) a light chain variable region CDR1 comprising the amino acid sequence of SEQ ID NO: 4; (v) a light chain variable region CDR2 comprising the amino acid sequence of SEQ ID NO: 5; and (vi) a light chain variable region CDR3 comprising the amino acid sequence of SEQ ID NO: 6.

The invention also comprises a combination comprising an anti-CD27 antibody or antigen binding fragment of the invention, in combination with one, two or more therapeutic agents; wherein the second therapeutic agent is selected from the group consisting of: an anti-LAG3 antibody or an antigen binding fragment thereof; an anti-TIGIT antibody or antigen binding fragment thereof; an anti-VISTA antibody or an antigen binding fragment thereof; an anti-BTLA antibody or an antigen binding fragment thereof; an anti-TIM3 antibody or an antigen binding fragment thereof; an anti-CTLA4 antibody or an antigen binding fragment thereof; an anti-HVEM antibody or an antigen binding fragment thereof; an anti-CD70 antibody or an antigen binding fragment thereof; an anti-OX40 antibody or an antigen binding fragment thereof; an anti-CD28 antibody or an antigen binding fragment thereof; an anti-PD1 antibody or an antigen binding fragment thereof; an anti-PDL1 antibody or an antigen binding fragment thereof; an anti-PDL2 antibody or an antigen binding fragment thereof; an anti-GITR antibody or an antigen binding fragment thereof; an anti-ICOS antibody or an antigen binding fragment thereof; an anti-SIRPα antibody or an antigen binding fragment thereof; an anti-ILT2 antibody or antigen binding fragment thereof; an anti-ILT3 antibody or antigen binding fragment thereof; an anti-ILT4 antibody or antigen binding fragment thereof; an anti-ILT5 antibody or an antigen binding fragment thereof; an anti 4-1BB antibody or an antigen binding fragment thereof; an anti-NKG2A antibody or an antigen binding fragment thereof; an anti-NKG2C antibody or an antigen binding fragment thereof; an anti-NKG2E antibody or an antigen binding fragment thereof; an anti-TSLP antibody or an antigen binding fragment thereof; an anti-IL-10 antibody or an antigen binding fragment thereof; IL-10 or PEGylated IL-10; a STING agonist; a CXCR2 antagonist; and a PARP inhibitor.

In one embodiment of the combinations of the invention, the anti-CD27 antibody or antigen binding fragment of the invention comprises a heavy chain variable region and a light chain variable region comprising: (i) a heavy chain variable region CDR1 comprising the amino acid sequence of SEQ ID NO: 1; (ii) a heavy chain variable region CDR2 comprising the amino acid sequence of SEQ ID NO: 2; (iii) a heavy chain variable region CDR3 comprising the amino acid sequence of SEQ ID NO: 3; (iv) a light chain variable region CDR1 comprising the amino acid sequence of SEQ ID NO: 4; (v) a light chain variable region CDR2 comprising the amino acid sequence of SEQ ID NO: 5; and (vi) a light chain variable region CDR3 comprising the amino acid sequence of SEQ ID NO: 6.

The invention also provides a vessel or injection device comprising any one of the anti-CD27 antibodies or antigen binding fragments of the invention. In one embodiment, the anti-CD27 antibody or antigen binding fragment of the invention comprises a heavy chain variable region and a light chain variable region comprising: (i) a heavy chain variable region CDR1 comprising the amino acid sequence of SEQ ID NO: 1; (ii) a heavy chain variable region CDR2 comprising the amino acid sequence of SEQ ID NO: 2; (iii) a heavy chain variable region CDR3 comprising the amino acid sequence of SEQ ID NO: 3; (iv) a light chain variable region CDR1 comprising the amino acid sequence of SEQ ID NO: 4; (v) a light chain variable region CDR2 comprising the amino acid sequence of SEQ ID NO: 5; and (vi) a light chain variable region CDR3 comprising the amino acid sequence of SEQ ID NO: 6. In another embodiment, the invention provides an antibody or antigen binding fragment that binds to human CD27 comprising a heavy chain variable region and a light chain variable region comprising: (i) a heavy chain variable region CDR1 comprising the amino acid sequence of SEQ ID NO: 1, wherein X₁=M; (ii) a heavy chain variable region CDR2 comprising the amino acid sequence of SEQ ID NO: 2, wherein X₁=N, X₂=T, X₃=N and X₄=T; (iii) a heavy chain variable region CDR3 comprising the amino acid sequence of SEQ ID NO: 3, wherein X₁=M; (iv) a light chain variable region CDR1 comprising the amino acid sequence of SEQ ID NO: 4, wherein X₁=M; (v) a light chain variable region CDR2 comprising the amino acid sequence of SEQ ID NO: 5, wherein X₁=D and X₂=T; and (vi) a light chain variable region CDR3 comprising the amino acid sequence of SEQ ID NO: 6, wherein X₁=W, X₂=N and X₃=S.

The invention also provides a method of producing an anti-CD27 antibody or antigen binding fragment of the invention comprising: culturing a host cell comprising a polynucleotide encoding a heavy chain and/or light chain of an antibody of the invention (or an antigen binding fragment thereof) under conditions favorable to expression of the polynucleotide; and optionally, recovering the antibody or antigen binding fragment from the host cell and/or culture medium. In one embodiment, the polynucleotide encoding the heavy chain and the polynucleotide encoding the light chain are in a single vector. In another embodiment, the polynucleotide encoding the heavy chain and the polynucleotide encoding the light chain are in different vectors. In one embodiment, the polynucleotide encoding the heavy chain and the polynucleotide encoding the light chain encode an antibody or antigen binding fragment comprising: (i) a heavy chain variable region CDR1 comprising the amino acid sequence of SEQ ID NO: 1; (ii) a heavy chain variable region CDR2 comprising the amino acid sequence of SEQ ID NO: 2; (iii) a heavy chain variable region CDR3 comprising the amino acid sequence of SEQ ID NO: 3; (iv) a light chain variable region CDR1 comprising the amino acid sequence of SEQ ID NO:4; (v) a light chain variable region CDR2 comprising the amino acid sequence of SEQ ID NO:5; and (vi) a light chain variable region CDR3 comprising the amino acid sequence of SEQ ID NO:6. In another embodiment, the invention provides a polynucleotide encoding a heavy chain and a polynucleotide encoding a light chain from an antibody or antigen binding fragment that binds to human CD27 comprising: (i) a heavy chain variable region CDR1 comprising the amino acid sequence of SEQ ID NO: 1, wherein X₁=M; (ii) a heavy chain variable region CDR2 comprising the amino acid sequence of SEQ ID NO: 2, wherein X₁=N, X₂=T, X₃=N and X₄=T; (iii) a heavy chain variable region CDR3 comprising the amino acid sequence of SEQ ID NO: 3, wherein X₁=M; (iv) a light chain variable region CDR1 comprising the amino acid sequence of SEQ ID NO: 4, wherein X₁=M; (v) a light chain variable region CDR2 comprising the amino acid sequence of SEQ ID NO: 5, wherein X₁=D and X₂=T; and (vi) a light chain variable region CDR3 comprising the amino acid sequence of SEQ ID NO: 6, wherein X₁=W, X₂=N and X₃=S.

The invention also provides a method of treating cancer in a subject in need thereof, comprising administering to the subject an effective amount of an anti-CD27 antibody or antigen binding fragment of the invention, optionally in association with a further therapeutic agent or therapeutic procedure. In one embodiment, the subject to be treated is a human subject. In one embodiment, the further therapeutic agent is selected from the group consisting of: an anti-LAG3 antibody or an antigen binding fragment thereof; an anti-TIGIT antibody or an antigen binding fragment thereof; an anti-VISTA antibody or an antigen binding fragment thereof; an anti-BTLA antibody or an antigen binding fragment thereof; an anti-TIM3 antibody or an antigen binding fragment thereof; an anti-CTLA4 antibody or an antigen binding fragment thereof; an anti-HVEM antibody or an antigen binding fragment thereof; an anti-CD70 antibody or an antigen binding fragment thereof; an anti-OX40 antibody or an antigen binding fragment thereof; an anti-CD28 antibody or an antigen binding fragment thereof; an anti-PD1 antibody or an antigen binding fragment thereof; an anti-PDL1 antibody or an antigen binding fragment thereof; an anti-PDL2 antibody or an antigen binding fragment thereof; an anti-GITR antibody or an antigen binding fragment thereof; an anti-ICOS antibody or an antigen binding fragment thereof; an anti-SIRPα antibody or an antigen binding fragment thereof; an anti-ILT2 antibody or antigen binding fragment thereof; an anti-ILT3 antibody or antigen binding fragment thereof; an anti-ILT4 antibody or antigen binding fragment thereof; an anti-ILT5 antibody or an antigen binding fragment thereof; and an anti-4-1BB antibody or an antigen binding fragment thereof; an anti-NKG2A antibody or an antigen binding fragment thereof; an anti-NKG2C antibody or an antigen binding fragment thereof; an anti-NKG2E antibody or an antigen binding fragment thereof; an anti-TSLP antibody or an antigen binding fragment thereof; an anti-IL-10 antibody or an antigen binding fragment thereof; an anti-IL-10 antibody or an antigen binding fragment thereof; IL-10 or PEGylated IL-10; a STING agonist; a CXCR2 antagonist; and a PARP inhibitor.

In one embodiment, the anti-CD27 antibody or antigen binding fragment of the invention comprises a heavy chain variable region and a light chain variable region comprising: (i) a heavy chain variable region CDR1 comprising the amino acid sequence of SEQ ID NO: 1; (ii) a heavy chain variable region CDR2 comprising the amino acid sequence of SEQ ID NO: 2; (iii) a heavy chain variable region CDR3 comprising the amino acid sequence of SEQ ID NO: 3; (iv) a light chain variable region CDR1 comprising the amino acid sequence of SEQ ID NO: 4; (v) a light chain variable region CDR2 comprising the amino acid sequence of SEQ ID NO: 5; and (vi) a light chain variable region CDR3 comprising the amino acid sequence of SEQ ID NO: 6. In another embodiment, the invention provides an antibody or antigen binding fragment that binds to human CD27 comprising a heavy chain variable region and a light chain variable region comprising: (i) a heavy chain variable region CDR1 comprising the amino acid sequence of SEQ ID NO: 1, wherein X₁=M; (ii) a heavy chain variable region CDR2 comprising the amino acid sequence of SEQ ID NO: 2, wherein X₁=N, X₂=T, X₃=N and X₄=T; (iii) a heavy chain variable region CDR3 comprising the amino acid sequence of SEQ ID NO: 3, wherein X₁=M; (iv) a light chain variable region CDR1 comprising the amino acid sequence of SEQ ID NO: 4, wherein X₁=M; (v) a light chain variable region CDR2 comprising the amino acid sequence of SEQ ID NO: 5, wherein X₁=D and X₂=T; and (vi) a light chain variable region CDR3 comprising the amino acid sequence of SEQ ID NO: 6, wherein X₁=W, X₂=N and X₃=S.

In one embodiment, the invention provides a composition comprising: (i) an anti-CD27 antibody or antigen binding fragment of the invention; and (ii) an anti-PD1 antibody comprising the heavy chain sequence of SEQ ID NO: 53 and the light chain sequence of SEQ ID NO: 48. In another embodiment, the invention provides a composition comprising: (a) an anti-CD27 antibody or antigen binding fragment of the invention; and (b) an anti-PD1 antibody comprising the heavy chain variable sequence of SEQ ID NO: 52 and the light chain variable sequence of SEQ ID NO: 78. In one embodiment, the anti-PD1 antibody is administered prior to the administration of an anti-CD27 antibody. In one embodiment, the anti-PD1 antibody is administered 4-10 days prior to the administration of the anti-CD27 antibody. In one embodiment, pretreatment with anti-PD1 antibody may modulate immune cells resulting in enhanced Fc-mediated function of the anti-CD27 antibodies. In one embodiment, the anti-CD27 antibody or antigen binding fragment of the invention comprises a heavy chain variable region and a light chain variable region comprising: (i) a heavy chain variable region CDR1 comprising the amino acid sequence of SEQ ID NO: 1; (ii) a heavy chain variable region CDR2 comprising the amino acid sequence of SEQ ID NO: 2; (iii) a heavy chain variable region CDR3 comprising the amino acid sequence of SEQ ID NO: 3; (iv) a light chain variable region CDR1 comprising the amino acid sequence of SEQ ID NO: 4; (v) a light chain variable region CDR2 comprising the amino acid sequence of SEQ ID NO: 5; and (vi) a light chain variable region CDR3 comprising the amino acid sequence of SEQ ID NO: 6.

The invention also provides a method of treating an infection or infectious disease in a subject, comprising administering to the subject an effective amount of an antibody or antigen binding fragment of the invention, optionally in association with a further therapeutic agent or therapeutic procedure. In one embodiment, the subject been treated is a human subject. In one embodiment, the further therapeutic agent is selected from the group consisting of: an anti-LAG3 antibody or an antigen binding fragment thereof; an anti-TIGIT antibody or an antigen binding fragment thereof; an anti-VISTA antibody or an antigen binding fragment thereof; an anti-BTLA antibody or an antigen binding fragment thereof; an anti-TIM3 antibody or an antigen binding fragment thereof; an anti-CTLA4 antibody or an antigen binding fragment thereof; an anti-HVEM antibody or an antigen binding fragment thereof; an anti-CD70 antibody or an antigen binding fragment thereof; an anti-OX40 antibody or an antigen binding fragment thereof; an anti-CD28 antibody or an antigen binding fragment thereof; an anti-PD1 antibody or an antigen binding fragment thereof; an anti-PDL1 antibody or an antigen binding fragment thereof; an anti-PDL2 antibody or an antigen binding fragment thereof; an anti-GITR antibody or an antigen binding fragment thereof; an anti-ICOS antibody or an antigen binding fragment thereof; an anti-SIRPα antibody or an antigen binding fragment thereof; an anti-ILT2 antibody or antigen binding fragment thereof; an anti-ILT3 antibody or antigen binding fragment thereof; an anti-ILT4 antibody or antigen binding fragment thereof; an anti-ILT5 antibody or an antigen binding fragment thereof; and an anti-4-1BB antibody or an antigen binding fragment thereof; an anti-NKG2A antibody or an antigen binding fragment thereof; an anti-NKG2C antibody or an antigen binding fragment thereof; an anti-NKG2E antibody or an antigen binding fragment thereof; an anti-TSLP antibody or an antigen binding fragment thereof; an anti-IL-10 antibody or an antigen binding fragment thereof; IL-10 or PEGylated IL-10; a STING agonist; a CXCR2 antagonist; and a PARP inhibitor.

In one embodiment, the anti-CD27 antibody or antigen binding fragment of the invention comprises a heavy chain variable region and a light chain variable region comprising: (i) a heavy chain variable region CDR1 comprising the amino acid sequence of SEQ ID NO: 1; (ii) a heavy chain variable region CDR2 comprising the amino acid sequence of SEQ ID NO: 2; (iii) a heavy chain variable region CDR3 comprising the amino acid sequence of SEQ ID NO: 3; (iv) a light chain variable region CDR1 comprising the amino acid sequence of SEQ ID NO: 4; (v) a light chain variable region CDR2 comprising the amino acid sequence of SEQ ID NO: 5; and (vi) a light chain variable region CDR3 comprising the amino acid sequence of SEQ ID NO: 6. In another embodiment, the invention provides an antibody or antigen binding fragment that binds to human CD27 comprising a heavy chain variable region and a light chain variable region comprising: (i) a heavy chain variable region CDR1 comprising the amino acid sequence of SEQ ID NO: 1, wherein X₁=M; (ii) a heavy chain variable region CDR2 comprising the amino acid sequence of SEQ ID NO: 2, wherein X₁=N, X₂=T, X₃=N and X₄=T; (iii) a heavy chain variable region CDR3 comprising the amino acid sequence of SEQ ID NO: 3, wherein X₁=M; (iv) a light chain variable region CDR1 comprising the amino acid sequence of SEQ ID NO: 4, wherein X₁=M; (v) a light chain variable region CDR2 comprising the amino acid sequence of SEQ ID NO: 5, wherein X₁=D and X₂=T; and (vi) a light chain variable region CDR3 comprising the amino acid sequence of SEQ ID NO: 6, wherein X₁=W, X₂=N and X₃=S.

The invention also provides a vaccine comprising an antibody or antigen binding fragment of the invention. In one embodiment, the anti-CD27 antibody or antigen binding fragment of the invention comprises a heavy chain variable region and a light chain variable region comprising: (i) a heavy chain variable region CDR1 comprising the amino acid sequence of SEQ ID NO: 1; (ii) a heavy chain variable region CDR2 comprising the amino acid sequence of SEQ ID NO: 2; (iii) a heavy chain variable region CDR3 comprising the amino acid sequence of SEQ ID NO: 3; (iv) a light chain variable region CDR1 comprising the amino acid sequence of SEQ ID NO: 4; (v) a light chain variable region CDR2 comprising the amino acid sequence of SEQ ID NO: 5; and (vi) a light chain variable region CDR3 comprising the amino acid sequence of SEQ ID NO: 6. In another embodiment, the invention provides an antibody or antigen binding fragment that binds to human CD27 comprising a heavy chain variable region and a light chain variable region comprising: (i) a heavy chain variable region CDR1 comprising the amino acid sequence of SEQ ID NO: 1, wherein X₁=M; (ii) a heavy chain variable region CDR2 comprising the amino acid sequence of SEQ ID NO: 2, wherein X₁=N, X₂=T, X₃=N and X₄=T; (iii) a heavy chain variable region CDR3 comprising the amino acid sequence of SEQ ID NO: 3, wherein X₁=M; (iv) a light chain variable region CDR1 comprising the amino acid sequence of SEQ ID NO: 4, wherein X₁=M; (v) a light chain variable region CDR2 comprising the amino acid sequence of SEQ ID NO: 5, wherein X₁=D and X₂=T; and (vi) a light chain variable region CDR3 comprising the amino acid sequence of SEQ ID NO: 6, wherein X₁=W, X₂=N and X₃=S. In one embodiment, the vaccine further comprises an antigen.

The invention also provides a method for detecting the presence of a CD27 peptide or a fragment thereof in a sample comprising contacting the sample with an antibody or antigen binding fragment thereof of the invention and detecting the presence of a complex between the antibody or fragment and the peptide; wherein detection of the complex indicates the presence of the CD27 peptide. In one embodiment, the anti-CD27 antibody or antigen binding fragment of the invention comprises: (i) a heavy chain variable region CDR1 comprising the amino acid sequence of SEQ ID NO: 1; (ii) a heavy chain variable region CDR2 comprising the amino acid sequence of SEQ ID NO: 2; (iii) a heavy chain variable region CDR3 comprising the amino acid sequence of SEQ ID NO: 3; (iv) a light chain variable region CDR1 comprising the amino acid sequence of SEQ ID NO: 4; (v) a light chain variable region CDR2 comprising the amino acid sequence of SEQ ID NO: 5; and (vi) a light chain variable region CDR3 comprising the amino acid sequence of SEQ ID NO: 6. In another embodiment, the invention provides an antibody or antigen binding fragment that binds to human CD27 comprising: (i) a heavy chain variable region CDR1 comprising the amino acid sequence of SEQ ID NO: 1, wherein X₁=M; (ii) a heavy chain variable region CDR2 comprising the amino acid sequence of SEQ ID NO: 2, wherein X₁=N, X₂=T, X₃=N and X₄=T; (iii) a heavy chain variable region CDR3 comprising the amino acid sequence of SEQ ID NO: 3, wherein X₁=M; (iv) a light chain variable region CDR1 comprising the amino acid sequence of SEQ ID NO: 4, wherein X₁=M; (v) a light chain variable region CDR2 comprising the amino acid sequence of SEQ ID NO: 5, wherein X₁=D and X₂=T; and (vi) a light chain variable region CDR3 comprising the amino acid sequence of SEQ ID NO: 6, wherein X₁=W, X₂=N and X₃=S.

The invention also provides a method of increasing the activity of an immune cell, comprising contacting the immune cell with any one of the antibodies or antigen binding fragments of the invention. In one embodiment, the invention provides a method of increasing the activity of an immune cell, comprising administering to a subject in need thereof an effective amount of an antibody or antigen binding fragments of the invention. In one embodiment, the method is used for: the treatment of cancer, the treatment of an infection or infectious disease, or as a vaccine adjuvant. In one embodiment, the increase in activity of an immune cell can be detected by measuring the proliferation of the immune cell. For example, an increase in activity of a T cell can be detected by measuring the proliferation of the T cell. In one embodiment, the increase in activity of an immune cell can be detected by measuring T cell activation ex vivo in a sample derived from the subject. In one embodiment, the increase in T cell activity is determined by: (i) measuring mixed lymphocyte reactions or direct anti-CD3 mAb stimulation of T cell receptor (TCR) signaling to induce production of a cytokine selected from the group consisting of: IL-2, TNFα, IL-17, IFNγ, IL-1β, GM-CSF, RANTES, IL-6, IL-8, IL-5 and IL-13; (ii) measuring SEB induced production of one or more cytokines selected from the group consisting of: IL-2, TNFα, IL-17, IFNγ, GM-CSF, RANTES, IL-6, IL-8, IL-5 and IL-13; or (iii) measuring TT induced production of a cytokine selected from the group consisting of: IL-2, TNFα, IL-17, IFNγ, GM-CSF, RANTES, IL-6, IL-8, IL-5 and IL-13. In one embodiment, the anti-CD27 antibody or antigen binding fragment of the invention comprises a heavy chain variable region and a light chain variable region comprising: (i) a heavy chain variable region CDR1 comprising the amino acid sequence of SEQ ID NO: 1; (ii) a heavy chain variable region CDR2 comprising the amino acid sequence of SEQ ID NO: 2; (iii) a heavy chain variable region CDR3 comprising the amino acid sequence of SEQ ID NO: 3; (iv) a light chain variable region CDR1 comprising the amino acid sequence of SEQ ID NO: 4; (v) a light chain variable region CDR2 comprising the amino acid sequence of SEQ ID NO: 5; and (vi) a light chain variable region CDR3 comprising the amino acid sequence of SEQ ID NO: 6. In another embodiment, the invention provides an antibody or antigen binding fragment that binds to human CD27 comprising a heavy chain variable region and a light chain variable region comprising: (i) a heavy chain variable region CDR1 comprising the amino acid sequence of SEQ ID NO: 1, wherein X₁=M; (ii) a heavy chain variable region CDR2 comprising the amino acid sequence of SEQ ID NO: 2, wherein X₁=N, X₂=T, X₃=N and X₄=T; (iii) a heavy chain variable region CDR3 comprising the amino acid sequence of SEQ ID NO: 3, wherein X₁=M; (iv) a light chain variable region CDR1 comprising the amino acid sequence of SEQ ID NO: 4, wherein X₁=M; (v) a light chain variable region CDR2 comprising the amino acid sequence of SEQ ID NO: 5, wherein X₁=D and X₂=T; and (vi) a light chain variable region CDR3 comprising the amino acid sequence of SEQ ID NO: 6, wherein X₁=W, X₂=N and X₃=S.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows that antibody hCD27.15 does not bind CD27 A59T. CD27 and CD27 A59T were expressed on CHO-K1 by transient transfection. Binding of hCD27.15 (humanized 6B) and Fc-mCD70 to CD27 and CD27 A59T were measured by flow cytometry. While Fc-mCD70 binds both CD27 and CD27 A59T, hCD27.15 only binds CD27.

FIG. 2 shows that antibody hCD27.15 does not bind Macaca mulatta CD27. HsCD27 and MmCD27 were expressed on CHO-K1 by transient transfection. Binding of hCD27.15 (chimeric c-4) to HsCD27 and MmCD27 was measured by flow cytometry.

FIG. 3A-3B shows an alignment of the heavy chain and light chain variable regions of various anti-CD27 antibodies of the invention to germline VH and VL sequences: 131AVH6 (SEQ ID NO: 10), 131AVH7 (SEQ ID NO: 11), 131A parental VH (SEQ ID NO: 7), 131AVH8 (SEQ ID NO: 12), 131AVH9 (SEQ ID NO: 13), VH1-102 (SEQ ID NO: 64), VH1-146 (SEQ ID NO: 65); 131AVL6 (SEQ ID NO: 15), 131AVL7 (SEQ ID NO: 16), 131A parental VL (SEQ ID NO: 8), 131AVL8 (SEQ ID NO: 17), 131AVL9 (SEQ ID NO: 18), VK1-O2 (SEQ ID NO: 66), VK3-L6 (SEQ ID NO: 67).

FIG. 4 shows anti-tumor activity of 131AVH6VL6-huIgG1 compared to 1F5-huIgG1 in huCD27 knock-in mice with MC38 tumors as measured by tumor volume and days post treatment.

FIG. 5 shows anti-tumor activity of 131AVH6VL6-huIgG1 in combination with anti-PD-1 in huCD27 knock-in mice with MB49 tumors.

FIGS. 6A-6C. hCD27.131AVH6VL6-huIgG1 induces NF-κB activation in CD27 expressing 293FT cells. Human embryonic kidney cells containing an NF-κB-Luciferase reporter construct (293FT-NF-κB-luciferase cells) were transiently transfected with plasmids encoding Human WT, A59T or Rhesus CD27. The cells were stimulated for 16-20 hours in the presence or absence of anti-human CD27 antibodies then assayed for NF-κB activation, as read out by luciferase activity. Shown are Fold Change values relative to unstimulated cells for luciferase activity after stimulation of (A) hCD27 WT, (B) hCD27 A59T, or (C) Rhesus-expressing HEK293FT cells with mAbs hCD27.131AVH6VHL6 huIgG1, 1F5 huIgG1, or hCD27.15-4B IgG4. Data represent triplicate measurements from 1 experiment (+SD). Data are representative of 3-6 independent experiments.

FIGS. 7A and 7B. hCD27.131A IgG1 and IgG4 humanization variants induce NF-κB activation in CD27 expressing 293FT cells. Human embryonic kidney cells containing an NF-κB-Luciferase reporter construct (293FT-NF-κB-luciferase cells) were transiently transfected with plasmid encoding human WT CD27. The cells were stimulated for 16-20 hours in presence or absence of anti-human CD27 antibodies then assayed for NF-κB activation, as read out by luciferase activity. Shown are Fold Change values relative to unstimulated cells for luciferase activity after stimulation with humanized hCD27.131A antibodies on (A) human IgG1 or (B) IgG4 frameworks.

FIG. 8. Bioactivity of 131AVH6VL6-IgG1 in Primary Human Tumor TIL Cultures. Human tumor tissues (N=20, 16 NSCLC, 3 RCC, and 1H&N) obtained from surgical resections were digested into a single cell suspension using the collagenase type I and DNase I. Enriched live cells containing a mixture of various cell types of tumor were treated with 0.1, 1, 10, or 20 μg/mL of 131AVH6VL6-hIgG1 in the presence of 10 ng/ml anti-CD3. Treatments also include the isotype control (IgG1 and IgG4, 20 μg/ml) and 10 μg/mL anti-PD-1 (pembrolizumab). Supernatants were collected for interferon gamma (IFNγ) measurement at Day 6. Shown are the mean IFNγ levels and the standard error of the mean. P-values for comparison between treatment groups and the isotype control groups were determined by the paired, parametric, two-tailed T-test. IFNγ=interferon gamma; IgG1=immunoglobulin G, subclass 1; IgG4=immunoglobulin G, subclass 4; SEM=standard error of the mean.

FIG. 9. Comparing Bioactivity of 131AVH6VL6-IgG1 with hCD27.15-4BIgG4 and IF5-IgG1 in Primary Human Tumor TIL Cultures. Digested human tumor cells from 13 tumors (12 NSCLC and 1 H&N cancer) of 20 were also tested with hCD27.15-4BIgG4 and IF5-IgG1 at the same time. Enriched live cells containing a mixture of various cell types of tumor were treated with 0.1, 1, 10, or 20 μg/mL of 131AVH6VL6-hIgG1, hCD27.15-4BIgG4, and IF5-IgG1, including the isotype control (IgG1 and IgG4, 20 μg/ml) and 10 μg/mL anti-PD1 (pembrolizumab) controls. 10 ng/ml soluble anti-CD3 (BioLegend, clone OKT3) was used as the stimulation. Supernatants were collected for interferon gamma (IFNγ) measurement at Day 6. Shown are the mean IFNγ levels and the standard error of the mean. P-values for comparison between treatment groups and the isotype control groups were determined by the paired, parametric, two-tailed T-test. IFNγ=interferon gamma; IgG1=immunoglobulin G, subclass 1; IgG4=immunoglobulin G, subclass 4; SEM=standard error of the mean.

FIG. 10. Binding of mouse hCD27.131A (mouse IgG1), humanized hCD27.15 (human IgG4, Clone 6B), 1A4 (Beckman Coulter IM2034, Clone 1A4CD27, mouse IgG1), 9F4 (Sanquin PeliCluster CD27; Art. No. M1455, Clone CLB-CD27/1, 9F4, mouse IgG2a) and 1F5IgG1 by flow cytometry to hCD27 alanine mutants expressed on CHO-K1 cells. The hCD27 variants that were tested are listed. Amino acids correspond to the UniProt P26842-1 sequence. Amino acids 1-20 constitute the signal peptide. Binding is expressed as the geometric mean of the FITC signal, relative to antibody binding to hCD27, which was set at 100%.

FIG. 11. Comparison of the structures of 131AVH6VL6Fab-CD27 and M2177Fab-CD27 complex (Obmolova et al. Mol Immunol. 2017 March; 83:92-99, 2017): the coordinates are shown as ribbon after superposition of the antigen structures; CD27 in black thick lines, and Fabs in thin lines, black, light grey for 131AVH6VL6Fab and M2177Fab, respectively. The coordinates for the M2177-CD27 complex are taken from PDB 5TLK using chains A, B and X for the Fab light chain, heavy chain, and antigen, respectively.

FIGS. 12A and 12B. Comparison of the structures of 131AVH6VL6Fab-CD27 (A) and M2177Fab-CD27 complex (B), close-up view: the input models and picture orientation are the same as for FIG. 11, but the view is zoomed on the Fab-antigen interface to better show the comparison. The same ribbon representation, color and thickness conventions are used as for FIG. 11 (except M2177-CD27 co-structure is colored light grey), but the side-chains of the residues involved in polar interactions are indicated as sticks. H-bonds and salt bridges with a distance cut-off of 3.30 Å are shown as dashed lines, using shorter dashes for salt bridges.

FIGS. 13A and 13B. Mouse anti-hCD27 clone hCD27.131A does not cross-compete with 1F5IgG1 for binding to hCD27 in cell-based ELISAs. Mouse hCD27.131A and mouse hCD27.15 were tested for competitive binding with 1F5IgG1 to hCD27. (A) Left panel: Experimental setup. Right panel: Cross-competition data of a serial dilution of 1F5IgG1 followed by 1 μg/ml mouse hCD27.131A, mouse hCD27.15 or mouse IgG1 isotype control. (B) Left panel: Experimental setup. Right panel: Cross-competition data of a serial dilution of mouse hCD27.131A, mouse hCD27.15 or mouse IgG1 isotype control, followed by 1 μg/ml 1F5IgG1. Data points with error bars represent mean of duplicate measurements with range.

FIG. 14. Bioactivity of 131AVH6VL6-hIgG1 and 1F5-hIgG1 in primary CD8 T cell co-stimulation assay.

DETAILED DESCRIPTION Abbreviations

Throughout the detailed description and examples of the invention the following abbreviations will be used:

-   ADCC Antibody-dependent cellular cytotoxicity -   CDC Complement-dependent cytotoxicity -   CDR Complementarity determining region in the immunoglobulin     variable regions, defined using the Kabat numbering system -   CHO Chinese hamster ovary -   ELISA Enzyme-linked immunosorbant assay -   FR Antibody framework region: the immunoglobulin variable regions     excluding the CDR regions. -   HRP Horseradish peroxidase -   IFN interferon -   IC50 concentration resulting in 50% inhibition -   IgG Immunoglobulin G -   Kabat An immunoglobulin alignment and numbering system pioneered by     Elvin A. Kabat ((1991) Sequences of Proteins of Immunological     Interest, 5th Ed. Public Health Service, National Institutes of     Health, Bethesda, Md.) -   mAb or Mab or MAb Monoclonal antibody -   SEB Staphylococcus Enterotoxin B -   TCR T cell receptor -   TT Tetanus toxoid -   V region The segment of Ig chains which is variable in sequence     between different antibodies. It extends to Kabat residue 107 in the     light chain and 113 in the heavy chain. -   VH Immunoglobulin heavy chain variable region -   VK Immunoglobulin kappa light chain variable region

Definitions

So that the invention may be more readily understood, certain technical and scientific terms are specifically defined below. Unless specifically defined elsewhere in this document, all other technical and scientific terms used herein have the meaning commonly understood by one of ordinary skill in the art to which this invention belongs.

As used herein, including the appended claims, the singular forms of words such as “a,” “an,” and “the,” include their corresponding plural references unless the context clearly dictates otherwise.

“Administration” and “treatment,” as applied to an animal, human, experimental subject, cell, tissue, organ, or biological fluid, refers to contact of an exogenous pharmaceutical, therapeutic, diagnostic agent, or composition to the animal, human, subject, cell, tissue, organ, or biological fluid. Treatment of a cell encompasses contact of a reagent to the cell, as well as contact of a reagent to a fluid, where the fluid is in contact with the cell. “Administration” and “treatment” also mean in vitro and ex vivo treatments, e.g., of a cell, by a reagent, diagnostic, binding compound, or by another cell.

“Treat” or “treating” means to administer a therapeutic agent, such as a composition containing any of the antibodies or antigen-binding fragments of the present invention, internally or externally to a subject or patient having one or more disease symptoms, or being suspected of having a disease, for which the agent has therapeutic activity. Typically, the agent is administered in an amount effective to alleviate one or more disease symptoms in the treated subject or population, whether by inducing the regression of or inhibiting the progression of such symptom(s) by any clinically measurable degree. The amount of a therapeutic agent that is effective to alleviate any particular disease symptom may vary according to factors such as the disease state, age, and weight of the patient, and the ability of the drug to elicit a desired response in the subject. Whether a disease symptom has been alleviated can be assessed by any clinical measurement typically used by physicians or other skilled healthcare providers to assess the severity or progression status of that symptom.

CD27

In an embodiment of the invention, the amino acid sequence of human CD27 comprises the amino acid sequence of SEQ ID NO: 19 or SEQ ID NO: 20 (which is identical to SEQ ID NO: 19 but includes a SNP-A59T. The frequency of the rs25680 allele (commonly referred to as A59T) has an overall average based on the ExAC database of 19.78%.

In an embodiment of the invention, the amino acid sequence of cynomolgus monkey, e.g., Macaca fascicularis CD27, or Macaca mulatta CD27 (mmCD27) comprises the amino acid sequence disclosed in SEQ ID NO: 21.

Anti-CD27 Antibodies and Antigen-Binding Fragments Thereof

The present invention provides antibodies or antigen-binding fragments thereof that bind human CD27 and uses of such antibodies or fragments. In some embodiments, the anti-CD27 antibodies are isolated.

In some embodiments, the anti-CD27 antibodies or antigen binding fragments of the invention bind to human CD27 (SEQ ID NO: 19 or SEQ ID NO: 20) with a KD of about 5-10 nM. In one embodiment, the antibody of the invention which binds to human CD27 is also cross-reactive with mmCD27. As used herein “cross-reactivity” refers to the ability of an antibody to react with a homologous protein from other species. Whether an antibody binds to human CD27 or mmCD27 can be determined using any assay known in the art. Examples of assays known in the art to determining binding affinity include surface plasmon resonance (e.g., BIACORE) or a similar technique (e.g. KinExa or OCTET).

The present invention includes anti-CD27 antibodies and methods of use thereof. As used herein, the term “antibody” refers to any form of antibody that exhibits the desired biological activity. Thus, it is used in the broadest sense and specifically covers, but is not limited to, monoclonal antibodies (including full length monoclonal antibodies comprising two light chains and two heavy chains), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), humanized antibodies, fully human antibodies, and chimeric antibodies.

The present invention includes anti-CD27 antigen-binding fragments and methods of use thereof. As used herein, unless otherwise indicated, “antibody fragment” or “antigen-binding fragment” refers to antigen-binding fragments of antibodies, i.e. antibody fragments that retain the ability to bind specifically to the antigen bound by the full-length antibody, e.g. fragments that retain one or more CDR regions. Examples of antigen-binding fragments include, but are not limited to, Fab, Fab′, F(ab′)₂, and Fv fragments; diabodies; linear antibodies; single-chain antibody molecules, e.g., sc-Fv; multispecific antibodies formed from antibody fragments.

The present invention includes anti-CD27 Fab fragments and methods of use thereof. A “Fab fragment” is comprised of one light chain and the C_(H)1 and variable regions of one heavy chain. The heavy chain of a Fab molecule cannot form a disulfide bond with another heavy chain molecule. A “Fab fragment” can be the product of papain cleavage of an antibody.

The present invention includes anti-CD27 antibodies and antigen-binding fragments thereof which comprise an Fc region and methods of use thereof. An “Fc” region contains two heavy chain fragments comprising the C_(H)3 and C_(H)2 domains of an antibody. The two heavy chain fragments are held together by two or more disulfide bonds and by hydrophobic interactions of the C_(H)3 domains.

The present invention includes anti-CD27 Fab′ fragments and methods of use thereof. A “Fab′ fragment” contains one light chain and a portion or fragment of one heavy chain that contains the VH domain and the C_(H)1 domain and also the region between the C_(H)1 and C_(H)2 domains, such that an interchain disulfide bond can be formed between the two heavy chains of two Fab′ fragments to form a F(ab′)₂ molecule.

The present invention includes anti-CD27 F(ab′)₂ fragments and methods of use thereof. A “F(ab′)₂ fragment” contains two light chains and two heavy chains containing a portion of the constant region between the C_(H)1 and C_(H)2 domains, such that an interchain disulfide bond is formed between the two heavy chains. A F(ab′)₂ fragment thus is composed of two Fab′ fragments that are held together by a disulfide bond between the two heavy chains. An “F(ab′)₂ fragment” can be the product of pepsin cleavage of an antibody.

The present invention includes anti-CD27 Fv fragments and methods of use thereof. The “Fv region” comprises the variable regions from both the heavy and light chains, but lacks the constant regions.

The present invention includes anti-CD27 scFv fragments and methods of use thereof. The term “single-chain Fv” or “scFv” antibody refers to antibody fragments comprising the V_(H) and V_(L) domains of an antibody, wherein these domains are present in a single polypeptide chain. Generally, the Fv polypeptide further comprises a polypeptide linker between the V_(H) and V_(L) domains which enables the scFv to form the desired structure for antigen-binding. For a review of scFv, see Pluckthun (1994) THE PHARMACOLOGY OF MONOCLONAL ANTIBODIES, vol. 113, Rosenburg and Moore eds. Springer-Verlag, New York, pp. 269-315. See also, International Patent Application Publication No. WO 88/01649 and U.S. Pat. Nos. 4,946,778 and 5,260,203.

The present invention includes anti-CD27 bivalent antibodies and methods of use thereof. A “bivalent antibody” comprises two antigen-binding sites. In some instances, the two binding sites have the same antigen specificities. However, bivalent antibodies may be bispecific (see below).

The present invention includes anti-CD27 diabodies and methods of use thereof. As used herein, the term “diabodies” refers to small antibody fragments with two antigen-binding sites, which fragments comprise a heavy chain variable domain (V_(H)) connected to a light chain variable domain (V_(L)) in the same polypeptide chain (V_(H)-V_(L) or V_(L)-V_(H)). By using a linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with the complementary domains of another chain and create two antigen-binding sites. Diabodies are described more fully in, e.g., EP 404,097; WO 93/11161; and Holliger et al. (1993) Proc. Natl. Acad. Sci. USA 90: 6444-6448. For a review of engineered antibody variants generally see Holliger and Hudson (2005) Nat. Biotechnol. 23:1126-1136.

Typically, an antibody or antigen-binding fragment of the invention which is modified in some way retains at least 10% of its binding activity (when compared to the parental antibody) when that activity is expressed on a molar basis. Preferably, an antibody or antigen-binding fragment of the invention retains at least 20%, 50%, 70%, 80%, 90%, 95% or 100% or more of the CD27 binding affinity as the parental antibody. It is also intended that an antibody or antigen-binding fragment of the invention can include conservative or non-conservative amino acid substitutions (referred to as “conservative variants” or “function conserved variants” of the antibody) that do not substantially alter its biologic activity.

The present invention includes isolated anti-CD27 antibodies and antigen-binding fragments thereof and methods of use thereof. “Isolated” antibodies or antigen-binding fragments thereof are at least partially free of other biological molecules from the cells or cell cultures in which they are produced. Such biological molecules include nucleic acids, proteins, lipids, carbohydrates, or other material such as cellular debris and growth medium. An isolated antibody or antigen-binding fragment may further be at least partially free of expression system components such as biological molecules from a host cell or of the growth medium thereof. Generally, the term “isolated” is not intended to refer to a complete absence of such biological molecules or to an absence of water, buffers, or salts or to components of a pharmaceutical formulation that includes the antibodies or fragments.

The present invention includes anti-CD27 chimeric antibodies (e.g., human constant domain/mouse variable domain) and methods of use thereof. As used herein, a “chimeric antibody” is an antibody having the variable domain from a first antibody and the constant domain from a second antibody, where the first and second antibodies are from different species. (U.S. Pat. No. 4,816,567; and Morrison et al., (1984) Proc. Natl. Acad. Sci. USA 81: 6851-6855). Typically, the variable domains are obtained from an antibody from an experimental animal (the “parental antibody”), such as a rodent, and the constant domain sequences are obtained from human antibodies, so that the resulting chimeric antibody will be less likely to elicit an adverse immune response in a human subject than the parental (e.g., mouse) antibody.

The present invention includes anti-CD27 humanized antibodies and antigen-binding fragments thereof (e.g., rat or mouse antibodies that have been humanized) and methods of use thereof. The invention includes any humanized version of the 131A antibody. As used herein “131A antibody” and “hCD27.131A” are used interchangeably to refer to an antibody comprising the V_(H) region of SEQ ID NO:7 and the V_(L) region of SEQ ID NO:8. As used herein, the term “humanized antibody” refers to forms of antibodies that contain sequences from both human and non-human (e.g., mouse or rat) antibodies. In general, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin, and all or substantially all of the framework (FR) regions are those of a human immunoglobulin sequence. The humanized antibody may optionally comprise at least a portion of a human immunoglobulin constant region (Fc). For more details about humanized antibodies, see, e.g., Jones et al., Nature, 321:522-525 (1986); Reichmann et al., Nature, 332:323-329 (1988); Presta, Curr. Op. Struct. Biol., 2:593-596 (1992); and Clark, Immunol. Today 21: 397-402 (2000).

In general, the basic antibody structural unit comprises a tetramer. Each tetramer includes two identical pairs of polypeptide chains, each pair having one “light” (about 25 kDa) and one “heavy” chain (about 50-70 kDa). The amino-terminal portion of each chain includes a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition. The carboxy-terminal portion of the heavy chain may define a constant region primarily responsible for effector function. Typically, human light chains are classified as kappa and lambda light chains. Furthermore, human heavy chains are typically classified as mu, delta, gamma, alpha, or epsilon, and define the antibody's isotype as IgM, IgD, IgG, IgA, and IgE, respectively. Within light and heavy chains, the variable and constant regions are joined by a “J” region of about 12 or more amino acids, with the heavy chain also including a “D” region of about 10 more amino acids. See generally, Fundamental Immunology Ch. 7 (Paul, W., ed., 2nd ed. Raven Press, N.Y. (1989).

The variable regions of each light/heavy chain pair form the antibody binding site. Thus, in general, an intact antibody has two binding sites. Except in bifunctional or bispecific antibodies, the two binding sites are, in general, the same.

Typically, the variable domains of both the heavy and light chains comprise three hypervariable regions, also called complementarity determining regions (CDRs), located within relatively conserved framework regions (FR). The CDRs are usually aligned by the framework regions, enabling binding to a specific epitope. In general, from N-terminal to C-terminal, both light and heavy chain variable domains comprise FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. The assignment of amino acids to each domain is, generally, in accordance with the definitions of Sequences of Proteins of Immunological Interest, Kabat, et al.; National Institutes of Health, Bethesda, Md.; 5th ed.; NIH Publ. No. 91-3242 (1991); Kabat (1978)Adv. Prot. Chem. 32:1-75; Kabat, et al., (1977) J. Biol. Chem. 252:6609-6616; Chothia, et al., (1987) J Mol. Biol. 196:901-917 or Chothia, et al., (1989) Nature 342:878-883.

As used herein, the term “framework” or “FR” residues refers to those variable domain residues other than the hypervariable region residues defined herein as CDR residues.

As used herein, the term “hypervariable region” refers to the amino acid residues of an antibody or antigen-binding fragment thereof that are responsible for antigen-binding. The hypervariable region comprises amino acid residues from a CDR (i.e. CDRL1, CDRL2 and CDRL3 in the light chain variable domain and CDRH1, CDRH2 and CDRH3 in the heavy chain variable domain). See Kabat et al. (1991) Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (defining the CDR regions of an antibody by sequence); see also Chothia and Lesk (1987) J. Mol. Biol. 196: 901-917 (defining the CDR regions of an antibody by structure).

“Isolated nucleic acid molecule” or “isolated polynucleotide” means a DNA or RNA of genomic, mRNA, cDNA, or synthetic origin or some combination thereof which is not associated with all or a portion of a polynucleotide in which the isolated polynucleotide is found in nature, or is linked to a polynucleotide to which it is not linked in nature. For purposes of this disclosure, it should be understood that “a nucleic acid molecule comprising” a particular nucleotide sequence does not encompass intact chromosomes. Isolated nucleic acid molecules “comprising” specified nucleic acid sequences may include, in addition to the specified sequences, coding sequences for up to ten or even up to twenty or more other proteins or portions or fragments thereof, or may include operably linked regulatory sequences that control expression of the coding region of the recited nucleic acid sequences, and/or may include vector sequences.

The phrase “control sequences” refers to DNA sequences necessary for the expression of an operably linked coding sequence in a particular host organism. The control sequences that are suitable for prokaryotes, for example, include a promoter, optionally an operator sequence, and a ribosome binding site. Eukaryotic cells are known to use promoters, polyadenylation signals, and enhancers.

A nucleic acid or polynucleotide is “operably linked” when it is placed into a functional relationship with another nucleic acid sequence. For example, DNA for a presequence or secretory leader is operably linked to DNA for a polypeptide if it is expressed as a preprotein that participates in the secretion of the polypeptide; a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation. Generally, but not always, “operably linked” means that the DNA sequences being linked are contiguous, and, in the case of a secretory leader, contiguous and in reading phase. However, enhancers do not have to be contiguous. Linking is accomplished by ligation at convenient restriction sites. If such sites do not exist, the synthetic oligonucleotide adaptors or linkers are used in accordance with conventional practice.

As used herein, the expressions “cell,” “cell line,” and “cell culture” are used interchangeably and all such designations include progeny. Thus, the words “transformants” and “transformed cells” include the primary subject cell and cultures derived therefrom without regard for the number of transfers. It is also understood that not all progeny will have precisely identical DNA content, due to deliberate or inadvertent mutations. Mutant progeny that have the same function or biological activity as screened for in the originally transformed cell are included. Where distinct designations are intended, it will be clear from the context.

As used herein, “germline sequence” refers to a sequence of unrearranged immunoglobulin DNA sequences. Any suitable source of unrearranged immunoglobulin sequences may be used. Human germline sequences may be obtained, for example, from JOINSOLVER germline databases on the website for the National Institute of Arthritis and Musculoskeletal and Skin Diseases of the United States National Institutes of Health. Mouse germline sequences may be obtained, for example, as described in Giudicelli et al. (2005) Nucleic Acids Res. 33:D256-D261.

Physical and Functional Properties of the Exemplary Anti-CD27 Antibodies

The present invention provides anti-CD27 antibodies and antigen-binding fragments thereof having specified structural and functional features, and methods of use of the antibodies or antigen-binding fragments thereof in the treatment or prevention of disease (e.g., cancer or infectious disease).

An “anti-CD27 antibody or antigen-binding fragment thereof of the present invention” includes: any antibody or antigen-binding fragment thereof that is discussed herein (e.g., hCD27.131A or humanized versions thereof disclosed in Table 12) or a variant thereof (e.g., sequence variant or functional variant); any antibody or antigen-binding fragment comprising any one or more of the CDRs set forth in Table 12.

As stated above, antibodies and fragments that bind to the same epitope as any of the anti-CD27 antibodies or antigen-binding fragments thereof of the present invention also form part of the present invention. In one embodiment, the invention provides an antibody or antigen binding fragment thereof that binds to the same epitope of human CD27 as an antibody comprising the variable heavy chain of SEQ ID NO: 10 and the variable light chain of SEQ ID NO: 15. In another embodiment, the invention provides an antibody or antigen binding fragment thereof that binds to the same epitope of human CD27 as an antibody comprising the variable heavy chain of SEQ ID NO:7 and the variable light chain of SEQ ID NO:8. There are several methods available for mapping antibody epitopes on target antigens, including: H/D-Ex Mass spec, X-ray crystallography, pepscan analysis, alanine scanning, hydroxyl radical footprinting and site directed mutagenesis. For example, HDX (Hydrogen Deuterium Exchange) coupled with proteolysis and mass spectrometry can be used to determine the epitope of an antibody on a specific antigen Y. HDX-MS relies on the accurate measurement and comparison of the degree of deuterium incorporation by an antigen when incubated in D₂O on its own and in presence of its antibody at various time intervals. Deuterium is exchanged with hydrogen on the amide backbone of the proteins in exposed areas whereas regions of the antigen bound to the antibody will be protected and will show less or no exchange after analysis by LC-MS/MS of proteolytic fragments. In one embodiment, the epitope is determined by solving the X-ray crystal structure of a complex between CD27 or fragment thereof and an anti-CD27 antibody or fragment thereof and identifying one or more CD27 residues within 4 A of the anti-CD27 antibody residues. In another embodiment, the epitope includes for example, CD27 residues that have van der Waals, polar interaction, salt bridge or hydrogen bond contact with the anti-CD27 antibody residues. In another embodiment, the epitope is determined by mutagenesis (for example Alanine scanning) of CD27 residues and analyzing the loss of binding to the anti-CD27 antibody as a result of the mutagenesis.

The invention provides an antibody or antigen binding fragment thereof, wherein, when bound to human CD27, binds at least one residue selected from the group consisting of Leu18, Asp34, Gln35, and Lys38 of SEQ ID NO: 19. In one embodiment, the antibody or antigen binding fragment thereof binds at least one, two, three or four residues selected from the group consisting of Leu18, Asp34, Gln35, and Lys38 of SEQ ID NO: 19. The invention also provides an antibody or antigen binding fragment thereof, wherein, when bound to human CD27, binds at least one residue selected from the group consisting of Gln35 and Lys38 of SEQ ID NO: 19. In one embodiment, the antibody or antigen binding fragment thereof binds at least Gln35 and Lys38 of SEQ ID NO: 19.

In another embodiment of the foregoing embodiments, the antibody or antigen binding fragment further binds one or more residues (one, two, three, four, five or six residues) selected from the group consisting of Pro8, Glu9, His11, Lys17, His36, and Arg37 of SEQ ID NO: 19. In a further embodiment, the antibody or antigen binding fragment further binds one or more residues (one, two, three, or four residues) selected from the group consisting of Glu9, Lys17, His36, and Arg37 of SEQ ID NO: 19. In another aspect of the invention, the antibody or antigen binding fragment thereof binds residues Pro8, Glu9, His11, Lys17, Leu18, Asp34, Gln35, His36, Arg37 and Lys38 of SEQ ID NO: 19. In a further embodiment, the antibody or antigen binding binds residues Glu9, Lys17, Leu18, Asp34, Gln35, His36, Arg37 and Lys38 of SEQ ID NO: 19.

In one embodiment of the foregoing embodiments, the antibody or antigen binding fragment thereof optionally has at least one of the following characteristics: binds to human CD27 with an EC₅₀ of less than 100 pM, or less than 200 pM according to Cell ELISA assay, binds to human CD27 A59T with an EC₅₀ of less than 150 pM, or less than 250 pM according to Cell ELISA assay; binds to rhesus CD27 with an EC₅₀ of less than 100 pM, or less than 150 pM according to Cell ELISA assay; binds to human CD27 and human CD27 (A59T) with a bivalent KD value of about 5-10 nM as determined by surface plasmon resonance (e.g., BIACORE) or a similar technique (e.g. KinExa or OCTET); cross-reacts with cynomolgus or rhesus CD27; blocks binding of human CD27 to human CD70; increases T cell activation; stimulates antigen-specific T-cell production of IL-2 and IFNγ; induces NF-κB activation in human CD27 expressing cells with an EC50 of less than 5 nM when the antibody or fragment thereof is in soluble form; induces NF-κB activation in human CD27A59T expressing cells with an EC50 of less than 10 nM when the antibody or fragment thereof is in soluble form; induces NF-κB activation in Rhesus CD27 expressing cells with an EC50 of less than 1 nM when the antibody or fragment thereof is in soluble form; has an EC50 of less than 0.5 nM for binding to human CD27 on CD8+ or CD4+ T cells; increases CD8+ T cell activation in soluble form; and increases anti-CD3-induced IFN_(γ) production in human tumor culture. In one embodiment, the human CD27, human CD27A59T, or rhesus CD27 expressing cells are HEK293FT human embryonic kidney cells containing an NF-κB-luciferase reporter construct with CD27 plasmids transiently transfected. In one embodiment, the CD8+ T cell activation is measured by the % CD8+ T cells that are CD25+CD69+, and there is an average of about 1.5-2-fold increase in CD8+ T cell activation. In another embodiment, the increase in anti-CD3-induced IFN_(γ) production is at least 1.5 fold at 20 ug/ml of anti-CD27 antibody, and 10 ng/ml of anti-CD3 antibody.

Examples of the immunoglobulin chains of anti-CD27 antibodies of the invention as well as their CDRs include, but are not limited those disclosed in Table 12 (SEQ ID NOs: 7-18 and 32-40). The present invention includes any polypeptide comprising or consisting of the amino acid sequences of SEQ ID NOs: 7-18 and 32-40, and 44-45, and recombinant nucleotides encoding such polypeptides.

The scope of the present invention includes isolated anti-CD27 antibodies and antigen-binding fragments thereof (e.g., humanized antibodies), comprising a variant of an immunoglobulin chain set forth herein, e.g., any of SEQ ID NOs:7-18, 32-40, and 44-45; wherein the variant exhibits one or more of the following properties: binds to human CD27 with an EC₅₀ of less than 100 pM, or less than 200 pM according to cell ELISA assay, binds to human CD27 A59T with an EC₅₀ of less than 150 pM, or less than 250 pM according to Cell ELISA assay; binds to rhesus CD27 with an EC₅₀ of less than 100 pM, or less than 150 pM according to Cell ELISA assay; binds to human CD27 and human CD27 (A59T) with a bivalent KD value of about 5-10 nM as determined by surface plasmon resonance (e.g., BIACORE) or a similar technique (e.g. KinExa or OCTET); cross-reacts with cynomolgus monkey or rhesus monkey CD27; blocks binding of human CD27 to human CD70; increases T cell activation; stimulates antigen-specific T-cell production of IL-2 and IFNγ; induces NF-κB activation in human CD27-expressing cells with an EC50 of less than 5 nM when the antibody or fragment thereof is in soluble form; induces NF-κB activation in human CD27A59T expressing cells with an EC50 of less than 10 nM when the antibody or fragment thereof is in soluble form; induces NF-κB activation in rhesus monkey CD27-expressing cells with an EC50 of less than 1 nM when the antibody or fragment thereof is in soluble form; has an EC50 of less than 0.5 nM for binding to human CD27 on CD8+ or CD4+ T cells; increases CD8+ T cell activation in soluble form; and increases anti-CD3-induced IFN, production in human tumor culture. In one embodiment, the human CD27, human CD27A59T, or rhesus CD27 expressing cells are HEK293FT human embryonic kidney cells containing an NF-κB-luciferase reporter construct with CD27 plasmids transiently transfected. In one embodiment, the CD8+ T cell activation is measured by the % CD8+ T cells that are CD25+CD69+, and there is an average of about 1.5-2-fold increase in CD8+ T cell activation. In another embodiment, the increase in anti-CD3-induced IFN_(γ) production is at least 1.5 fold at 20 ug/ml of anti-CD27 antibody, and 10 ng/ml of anti-CD3 antibody. In one embodiment, the variant comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 amino acid substitutions with respect to any one of SEQ ID NOs: 7-18, 32-40 and 44-45.

In other embodiments, the invention provides antibodies or antigen-binding fragment thereof that bind human CD27 (e.g., humanized antibodies) and have V_(L) domains and V_(H) domains with at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity with at least one of SEQ ID NOs: 7-18 and 32-40; wherein the variant exhibits the desired binding and properties, e.g., binds to human CD27 with an EC₅₀ of less than 100 pM, or less than 200 pM according to cell ELISA assay, binds to human CD27 A59T with an EC₅₀ of less than 150 pM, or less than 250 pM according to cell ELISA assay; binds to rhesus monkey CD27 with an EC₅₀ of less than 100 pM, or less than 150 pM according to cell ELISA assay; binds to human CD27 and human CD27 (A59T) with a bivalent KD value of about 5-10 nM as determined by surface plasmon resonance (e.g., BIACORE) or a similar technique (e.g. KinExa or OCTET); cross-reacts with cynomolgus monkey or rhesus monkey CD27; blocks binding of human CD27 to human CD70; increases T cell activation; stimulates antigen-specific T-cell production of IL-2 and IFNγ; induces NF-κB activation in human CD27-expressing cells with an EC50 of less than 5 nM when the antibody or fragment thereof is in soluble form; induces NF-κB activation in human CD27A59T-expressing cells with an EC50 of less than 10 nM when the antibody or fragment thereof is in soluble form; induces NF-κB activation in rhesus monkey CD27-expressing cells with an EC50 of less than 1 nM when the antibody or fragment thereof is in soluble form; has an EC50 of less than 0.5 nM for binding to human CD27 on CD8+ or CD4+ T cells; increases CD8+ T cell activation in soluble form; and increases anti-CD3-induced IFN_(γ) production in human tumor culture. In one embodiment, the human CD27, human CD27A59T, or rhesus CD27 expressing cells are HEK293FT human embryonic kidney cells containing an NF-κB-luciferase reporter construct with CD27 plasmids transiently transfected. In one embodiment, the CD8+ T cell activation is measured by the % CD8+ T cells that are CD25+CD69+, and there is an average of about 1.5-2-fold increase in CD8+ T cell activation. In another embodiment, the increase in anti-CD3-induced IFN_(γ) production is at least 1.5 fold at 20 ug/ml of anti-CD27 antibody, and 10 ng/ml of anti-CD3 antibody.

In other embodiments, the invention provides antibodies or antigen-binding fragments thereof that bind human CD27 (e.g., humanized antibodies) and have V_(L) domains and V_(H) domains with at least 95% sequence identity with any one of the V_(L) domains of SEQ ID NOs:8, 14-18, 33, 35 and 40, and any one of the V_(H) domains of SEQ ID NOs: 7, 9-13, 32, 34, and 39. In other embodiments, the invention provides antibodies or antigen-binding fragment thereof that bind human CD27 (e.g., humanized antibodies) and have V_(L) domains and V_(H) domains with any one of the V_(L) domains of SEQ ID NOs:8, 14-18, 33, 35 and 40, and any one of the V_(H) domains of SEQ ID NOs: 7, 9-13, 32, 34, and 39. In other embodiments, the invention provides antibodies or antigen-binding fragment thereof that bind human CD27 (e.g., humanized antibodies) and have V_(L) domains and V_(H) domains with at least 99% sequence identity with any one of the V_(L) domains of SEQ ID NOs:8, 14-18, 33, 35 and 40, and any one of the V_(H) domains of SEQ ID NOs: 7, 9-13, 32, 34, and 39.

“Conservatively modified variants” or “conservative substitution” refers to substitutions of amino acids in a protein with other amino acids having similar characteristics (e.g. charge, side-chain size, hydrophobicity/hydrophilicity, backbone conformation and rigidity, etc.), such that the changes can frequently be made without altering the biological activity of the protein. Those of skill in this art recognize that, in general, single amino acid substitutions in non-essential regions of a polypeptide do not substantially alter biological activity (see, e.g., Watson et al. (1987) Molecular Biology of the Gene, The Benjamin/Cummings Pub. Co., p. 224 (4th Ed.)). In addition, substitutions of structurally or functionally similar amino acids are less likely to disrupt biological activity. Exemplary conservative substitutions are set forth in Table 1.

TABLE 1 Exemplary Conservative Amino Acid Substitutions Original residue Conservative substitution Ala (A) Gly; Ser Arg (R) Lys; His Asn (N) Gln; His Asp (D) Glu; Asn Cys (C) Ser; Ala Gln (Q) Asn Glu (E) Asp; Gln Gly (G) Ala His (H) Asn; Gln Ile (I) Leu; Val Leu (L) Ile; Val Lys (K) Arg; His Met (M) Leu; Ile; Tyr Phe (F) Tyr; Met; Leu Pro (P) Ala Ser (S) Thr Thr (T) Ser Trp (W) Tyr; Phe Tyr (Y) Trp; Phe Val (V) Ile; Leu

Function-conservative variants of the antibodies of the invention are also contemplated by the present invention. “Function-conservative variants,” as used herein, refers to antibodies or fragments in which one or more amino acid residues have been changed without altering a desired property, such as an antigen affinity and/or specificity. Such variants include, but are not limited to, replacement of an amino acid with one having similar properties, such as the conservative amino acid substitutions of Table 1. Also provided are isolated polypeptides comprising the V_(L) domains of the anti-CD27 antibodies of the invention (e.g., SEQ ID NOs: 8, 14-18, 33, 35 and 40), and isolated polypeptides comprising the V_(H) domains of the anti-CD27 antibodies of the invention (e.g., SEQ ID NOs: 7, 9-13, 32, 34, and 39) having up to 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acid substitutions.

In another embodiment, provided is an antibody or antigen-binding fragment thereof that binds CD27 and has V_(L) domains and V_(H) domains with at least 99% 98%, 97%, 96%, 95%, 90%, 85%, 80% or 75% sequence identity to one or more of the V_(L) domains or V_(H) domains described herein, and exhibits specific binding to CD27. In another embodiment the binding antibody or antigen-binding fragment thereof of the present invention comprises V_(L) and V_(H) domains (with and without signal sequence) having up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or more amino acid substitutions, and exhibits specific binding to CD27.

Polynucleotides and Polypeptides

The present invention further comprises polynucleotides encoding any of the polypeptides or immunoglobulin chains of anti-CD27 antibodies and antigen-binding fragments thereof of the invention. For example, the present invention includes the polynucleotides encoding the amino acids described in any one of SEQ ID NOs: 1-18, 32-40, and 44-45. In another embodiment, the invention provides an isolated nucleic acid comprising SEQ ID NO: 46 or SEQ ID NO: 47, or both.

In one embodiment, an isolated polynucleotide, for example DNA, encoding the polypeptide chains of the isolated antibodies or antigen-binding fragments set forth herein is provided. In one embodiment, the isolated polynucleotide encodes an antibody or antigen-binding fragment thereof comprising at least one mature immunoglobulin light chain variable (V_(L)) domain according to the invention and/or at least one mature immunoglobulin heavy chain variable (V_(H)) domain according to the invention. In some embodiments the isolated polynucleotide encodes both a light chain and a heavy chain on a single polynucleotide molecule, and in other embodiments the light and heavy chains are encoded on separate polynucleotide molecules. In another embodiment the polynucleotides further encodes a signal sequence.

In one embodiment, the invention comprises an isolated polynucleotide encoding a V_(H) domain or an antigen-binding fragment thereof comprising CDR-H1 (SEQ ID NO: 1), CDR-H2 (SEQ ID NO:2) and CDR-H3 (SEQ ID NO:3).

In one embodiment, the invention comprises an isolated polynucleotide encoding a V_(L) domain or an antigen-binding fragment thereof comprising CDR-L1 (SEQ ID NO:4), CDR-L2 (SEQ ID NO:5) and CDR-L3 (SEQ ID NO:6).

In one embodiment, the invention comprises an isolated polynucleotide encoding the V_(H) domain of SEQ ID NO: 7.

In one embodiment, the invention comprises an isolated polynucleotide encoding the V_(L) domain of SEQ ID NO: 8.

In one embodiment, the invention comprises an isolated polynucleotide encoding the V_(H) domain of any one of SEQ ID NOs: 10-13.

In one embodiment, the invention comprises an isolated polynucleotide encoding the V_(H) domain of SEQ ID NO: 10.

In one embodiment, the invention comprises an isolated polynucleotide encoding the V_(L) domain of any one of SEQ ID NOs: 15-18.

In one embodiment, the invention comprises an isolated polynucleotide encoding the V_(L) domain of SEQ ID NO: 15.

This present invention also provides vectors, e.g., expression vectors, such as plasmids, comprising the isolated polynucleotides of the invention, wherein the polynucleotide is operably linked to control sequences that are recognized by a host cell when the host cell is transfected with the vector. Also provided are host cells comprising a vector of the present invention and methods for producing the antibody or antigen-binding fragment thereof or polypeptide disclosed herein comprising culturing a host cell harboring an expression vector or a nucleic acid encoding the immunoglobulin chains of the antibody or antigen-binding fragment thereof in culture medium, and isolating the antigen or antigen-binding fragment thereof from the host cell or culture medium.

Also included in the present invention are polypeptides, e.g., immunoglobulin polypeptides, comprising amino acid sequences that are at least about 75% identical, 80% identical, more preferably at least about 90% identical and most preferably at least about 95% identical (e.g., 95%, 96%, 97%, 98%, 99%, 100%) to the amino acid sequences of the antibodies provided herein when the comparison is performed by a BLAST algorithm wherein the parameters of the algorithm are selected to give the largest match between the respective sequences over the entire length of the respective reference sequences (e.g. expect threshold: 10; word size: 3; max matches in a query range: 0; BLOSUM 62 matrix; gap costs: existence 11, extension 1; conditional compositional score matrix adjustment).

Sequence identity refers to the degree to which the amino acids of two polypeptides are the same at equivalent positions when the two sequences are optimally aligned.

The following references relate to BLAST algorithms often used for sequence analysis: BLAST ALGORITHMS: Altschul et al. (2005) FEBS J. 272(20): 5101-5109; Altschul, S. F., et al., (1990) J. Mol. Biol. 215:403-410; Gish, W., et al., (1993) Nature Genet. 3:266-272; Madden, T. L., et al., (1996) Meth. Enzymol. 266:131-141; Altschul, S. F., et al., (1997) Nucleic Acids Res. 25:3389-3402; Zhang, J., et al., (1997) Genome Res. 7:649-656; Wootton, J. C., et al., (1993) Comput. Chem. 17:149-163; Hancock, J. M. et al., (1994) Comput. Appl. Biosci. 10:67-70; ALIGNMENT SCORING SYSTEMS: Dayhoff, M. O., et al., “A model of evolutionary change in proteins.” in Atlas of Protein Sequence and Structure, (1978) vol. 5, suppl. 3. M. O. Dayhoff (ed.), pp. 345-352, Natl. Biomed. Res. Found., Washington, D.C.; Schwartz, R. M., et al., “Matrices for detecting distant relationships.” in Atlas of Protein Sequence and Structure, (1978) vol. 5, suppl. 3.” M. O. Dayhoff (ed.), pp. 353-358, Natl. Biomed. Res. Found., Washington, D.C.; Altschul, S. F., (1991) J. Mol. Biol. 219:555-565; States, D. J., et al., (1991) Methods 3:66-70; Henikoff, S., et al., (1992) Proc. Natl. Acad. Sci. USA 89:10915-10919; Altschul, S. F., et al., (1993) J. Mol. Evol. 36:290-300; ALIGNMENT STATISTICS: Karlin, S., et al., (1990) Proc. Natl. Acad. Sci. USA 87:2264-2268; Karlin, S., et al., (1993) Proc. Natl. Acad. Sci. USA 90:5873-5877; Dembo, A., et al., (1994) Ann. Prob. 22:2022-2039; and Altschul, S. F. “Evaluating the statistical significance of multiple distinct local alignments.” in Theoretical and Computational Methods in Genome Research (S. Suhai, ed.), (1997) pp. 1-14, Plenum, New York.

Binding Affinity

By way of example, and not limitation, the antibodies and antigen-binding fragments disclosed herein may bind human CD27 or CD27A59T (SEQ ID NO: 19 or SEQ ID NO: 20) with a bivalent KD value of 10×10⁻⁹ M or lower as determined by surface plasmon resonance (e.g., BIACORE) or a similar technique (e.g. KinExa or OCTET) as measured with a human CD27-Fc fusion protein or human CD27A59T-Fc fusion protein. In one embodiment, the antibodies and antigen-binding fragments disclosed herein may bind human CD27 or CD27A59T with a bivalent KD value of about 5-10×10⁻⁹ M as determined by surface plasmon resonance (e.g., BIACORE) or a similar technique (e.g. KinExa or OCTET) as measured with a human CD27-Fc fusion protein or human CD27A59T-Fc fusion protein.

Immune Cell Activation

In some embodiments, the antibodies or antigen binding fragments of the invention increase the activity of an immune cell. The increase of the activity of an immune cell can be detected using any method known in the art. In one embodiment, the increase in activity of an immune cell can be detected by measuring the proliferation of the immune cell. For example, an increase in activity of a T cell can be detected by measuring the proliferation of the T cell or signal transduction events such as tyrosine phosphorylation of immune receptors or downstream kinases that transmit signals to transcriptional regulators. In other embodiments, the increase in activity of an immune cell can be detected by measuring CTL or NK cell cytotoxic function on specific target cells or IFNγ cytokine responses, which are associated with stimulation of anti-tumor immunity. In yet other embodiments, the increase in activity of an immune cell can be detected by measuring T cell activation ex vivo in a sample derived from the subject. In one embodiment, the increase in T cell activity is determined by: (i) measuring SEB (Staphylococcus Enterotoxin B) induced production of one or more pro-inflammatory cytokines selected from the group consisting of: IL-2, TNFα, IL-17, IFNγ, IL-1β, GM-CSF, RANTES, IL-6, IL-8, IL-5 and IL-13; or (ii) measuring mixed lymphocyte reactions or direct anti-CD3 mAb stimulation of TCR signaling to induce production of a cytokine selected from the group consisting of: IL-2, TNFα, IL-17, IFNγ, IL-1β, GM-CSF, RANTES, IL-6, IL-8, IL-5 and IL-13. In certain embodiments, the anti-CD27 antibody or antigen binding fragment thereof of the present invention will stimulate antigen-specific T-cell production of IL-2 and/or IFNγ by at least 1.5 fold.

In some embodiments, the ability of the antibodies or antigen binding fragments of the invention to increase the activity of an immune cell can be detected by CD25 and CD69 upregulation by flow cytometry.

Ability of Anti-hCD27 Antibodies to Block Binding to hCD70

In some embodiments, the anti-CD27 antibodies or antigen binding fragments of the invention are able to block binding of human CD27 to human CD70. The ability to block binding of human CD27 to human CD70 can be determined using any method known in the art. In one embodiment, the ability of the antibodies to block binding of human CD27 to human CD70 is determined using an ELISA assay.

Methods of Making Antibodies and Antigen-Binding Fragments Thereof

Thus, the present invention includes methods for making an anti-CD27 antibody or antigen-binding fragment thereof of the present invention comprising culturing a hybridoma cell that expresses the antibody or fragment under conditions favorable to such expression and, optionally, isolating the antibody or fragment from the hybridoma and/or the growth medium (e.g. cell culture medium).

The anti-CD27 antibodies disclosed herein may also be produced recombinantly (e.g., in an E. coli/T7 expression system, a mammalian cell expression system or a lower eukaryote expression system). In this embodiment, nucleic acids encoding the antibody immunoglobulin molecules of the invention (e.g., V_(H) or V_(L)) may be inserted into a pET-based plasmid and expressed in the E. coli/T7 system. For example, the present invention includes methods for expressing an antibody or antigen-binding fragment thereof or immunoglobulin chain thereof in a host cell (e.g., bacterial host cell such as E. coli such as BL21 or BL21DE3) comprising expressing T7 RNA polymerase in the cell which also includes a polynucleotide encoding an immunoglobulin chain that is operably linked to a T7 promoter. For example, in an embodiment of the invention, a bacterial host cell, such as a E. coli, includes a polynucleotide encoding the T7 RNA polymerase gene operably linked to a lac promoter and expression of the polymerase and the chain is induced by incubation of the host cell with IPTG (isopropyl-beta-D-thiogalactopyranoside).

There are several methods by which to produce recombinant antibodies which are known in the art. One example of a method for recombinant production of antibodies is disclosed in U.S. Pat. No. 4,816,567.

Transformation can be by any known method for introducing polynucleotides into a host cell. Methods for introduction of heterologous polynucleotides into mammalian cells are well known in the art and include dextran-mediated transfection, calcium phosphate precipitation, polybrene-mediated transfection, protoplast fusion, electroporation, encapsulation of the polynucleotide(s) in liposomes, biolistic injection and direct microinjection of the DNA into nuclei. In addition, nucleic acid molecules may be introduced into mammalian cells by viral vectors. Methods of transforming cells are well known in the art. See, for example, U.S. Pat. Nos. 4,399,216; 4,912,040; 4,740,461 and 4,959,455.

Thus, the present invention includes recombinant methods for making an anti-CD27 antibody or antigen-binding fragment thereof of the present invention, or an immunoglobulin chain thereof, comprising introducing a polynucleotide encoding one or more immunoglobulin chains of the antibody or fragment (e.g., heavy and/or light immunoglobulin chain); culturing the host cell (e.g., CHO or Pichia or Pichia pastoris) under condition favorable to such expression and, optionally, isolating the antibody or fragment or chain from the host cell and/or medium in which the host cell is grown.

Anti-CD27 antibodies can also be synthesized by any of the methods set forth in U.S. Pat. No. 6,331,415.

Eukaryotic and prokaryotic host cells, including mammalian cells as hosts for expression of the antibodies or fragments or immunoglobulin chains disclosed herein are well known in the art and include many immortalized cell lines available from the American Type Culture Collection (ATCC). These include, inter alia, Chinese hamster ovary (CHO) cells, NSO, SP2 cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells (e.g., Hep G2), A549 cells, 3T3 cells, HEK-293 cells and a number of other cell lines. Mammalian host cells include human, mouse, rat, dog, monkey, pig, goat, bovine, horse and hamster cells. Cell lines of particular preference are selected through determining which cell lines have high expression levels. Other cell lines that may be used are insect cell lines, such as Sf9 cells, amphibian cells, bacterial cells, plant cells and fungal cells. Fungal cells include yeast and filamentous fungus cells including, for example, Pichia pastoris, Pichia finlandica, Pichia trehalophila, Pichia koclamae, Pichia membranaefaciens, Pichia minuta (Ogataea minuta, Pichia lindneri), Pichia opuntiae, Pichia thermotolerans, Pichia salictaria, Pichia guercuum, Pichia pijperi, Pichia stiptis, Pichia methanolica, Pichia sp., Saccharomyces cerevisiae, Saccharomyces sp., Hansenula polymorpha, Kluyveromyces sp., Kluyveromyces lactis, Candida albicans, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Trichoderma reesei, Chrysosporium lucknowense, Fusarium sp., Fusarium gramineum, Fusarium venenatum, Physcomitrella patens and Neurospora crassa. Pichia sp., any Saccharomyces sp., Hansenula polymorpha, any Kluyveromyces sp., Candida albicans, any Aspergillus sp., Trichoderma reesei, Chrysosporium lucknowense, any Fusarium sp., Yarrowia lipolytica, and Neurospora crassa. When recombinant expression vectors encoding the heavy chain or antigen-binding portion or fragment thereof, the light chain and/or antigen-binding fragment thereof are introduced into mammalian host cells, the antibodies are produced by culturing the host cells for a period of time sufficient to allow for expression of the antibody or fragment or chain in the host cells or secretion of the into the culture medium in which the host cells are grown.

Antibodies and antigen-binding fragments thereof and immunoglobulin chains can be recovered from the culture medium using standard protein purification methods. Further, expression of antibodies and antigen-binding fragments thereof and immunoglobulin chains of the invention (or other moieties therefrom) from production cell lines can be enhanced using a number of known techniques. For example, the glutamine synthetase gene expression system (the GS system) is a common approach for enhancing expression under certain conditions. The GS system is discussed in whole or part in connection with European Patent Nos. 0 216 846, 0 256 055, and 0 323 997 and European Patent Application No. 89303964.4. Thus, in an embodiment of the invention, the mammalian host cells (e.g., CHO) lack a glutamine synthetase gene and are grown in the absence of glutamine in the medium wherein, however, the polynucleotide encoding the immunoglobulin chain comprises a glutamine synthetase gene which complements the lack of the gene in the host cell.

In general, glycoproteins produced in a particular cell line or transgenic animal will have a glycosylation pattern that is characteristic for glycoproteins produced in the cell line or transgenic animal. Therefore, the particular glycosylation pattern of an antibody will depend on the particular cell line or transgenic animal used to produce the antibody. However, all antibodies encoded by the nucleic acid molecules provided herein, or comprising the amino acid sequences provided herein, comprise the instant invention, independent of the glycosylation pattern the antibodies may have. Similarly, in particular embodiments, antibodies with a glycosylation pattern comprising only non-fucosylated N-glycans may be advantageous, because these antibodies have been shown to typically exhibit more potent efficacy than their fucosylated counterparts both in vitro and in vivo (See for example, Shinkawa et al., J. Biol. Chem. 278: 3466-3473 (2003); U.S. Pat. Nos. 6,946,292 and 7,214,775). These antibodies with non-fucosylated N-glycans are not likely to be immunogenic because their carbohydrate structures are a normal component of the population that exists in human serum IgG.

The present invention includes bispecific and bifunctional antibodies and antigen-binding fragments having a binding specificity for CD27 and another antigen such as, for example, PD-1, PD-L1 or LAG-3, and methods of use thereof. In an embodiment of the invention, the anti-CD27 chains comprise any one of the VH/VL sequences described in Table 12, and the anti-PD1 chains comprise the amino acid sequence of SEQ ID NOs: 48 and 53 or of SEQ ID NOs: 78 and 52 (or an antigen binding fragment of any of said sequences). A bispecific or bifunctional antibody is an artificial hybrid antibody having two different heavy/light chain pairs and two different binding sites. Bispecific antibodies can be produced by a variety of methods including fusion of hybridomas or linking of Fab′ fragments. See, e.g., Songsivilai, et al., (1990) Clin. Exp. Immunol. 79: 315-321, Kostelny, et al., (1992)J Immunol. 148:1547-1553. In addition, bispecific antibodies may be formed as “diabodies” (Holliger, et al., (1993) PNAS USA 90:6444-6448) or as “Janusins” (Traunecker, et al., (1991) EMBO J 10:3655-3659 and Traunecker, et al., (1992) Int. J. Cancer Suppl. 7:51-52).

The present invention further includes anti-CD27 antigen-binding fragments of the anti-CD27 antibodies disclosed herein. The antibody fragments include F(ab)₂ fragments, which may be produced by enzymatic cleavage of an IgG by, for example, pepsin. Fab fragments may be produced by, for example, reduction of F(ab)₂ with dithiothreitol or mercaptoethylamine.

Immunoglobulins may be assigned to different classes depending on the amino acid sequences of the constant domain of their heavy chains. In some embodiments, different constant domains may be appended to humanized V_(L) and V_(H) regions derived from the CDRs provided herein. There are at least five major classes of immunoglobulins: IgA, IgD, IgE, IgG and IgM, and several of these may be further divided into subclasses (isotypes), e.g. IgG1, IgG2, IgG3 and IgG4; IgA1 and IgA2. The invention comprises antibodies and antigen-binding fragments of any of these classes or subclasses of antibodies.

In one embodiment, the antibody or antigen-binding fragment comprises a heavy chain constant region, e.g. a human constant region, such as γ1, γ2, γ3, or γ4 human heavy chain constant region or a variant thereof. In another embodiment, the antibody or antigen-binding fragment comprises a light chain constant region, e.g. a human light chain constant region, such as lambda or kappa human light chain region or variant thereof. By way of example, and not limitation the human heavy chain constant region can be γ4 and the human light chain constant region can be kappa. In an alternative embodiment, the Fc region of the antibody is γ4 with a Ser228Pro mutation (Schuurman, J et. al., Mol. Immunol. 38: 1-8, 2001).

In one embodiment, the antibody or antigen-binding fragment comprises a heavy chain constant region of the IgG1 subtype. In one embodiment, the antibody or antigen-binding fragment comprises a heavy chain constant region of the IgG2 subtype. In one embodiment, the antibody or antigen-binding fragment comprises a heavy chain constant region of the IgG4 subtype.

Antibody Engineering

Further included are embodiments in which the anti-CD27 antibodies and antigen-binding fragments thereof are engineered antibodies to include modifications to framework residues within the variable domains of the parental hCD27.131A monoclonal antibody, e.g. to improve the properties of the antibody or fragment. Typically, such framework modifications are made to decrease the immunogenicity of the antibody or fragment. This is usually accomplished by replacing non-CDR residues in the variable domains (i.e. framework residues) in a parental (e.g. rodent) antibody or fragment with analogous residues from the immune repertoire of the species in which the antibody is to be used, e.g. human residues in the case of human therapeutics. Such an antibody or fragment is referred to as a “humanized” antibody or fragment. In some cases it is desirable to increase the affinity, or alter the specificity of an engineered (e.g. humanized) antibody. One approach is to “backmutate” one or more framework residues to the corresponding germline sequence. More specifically, an antibody or fragment that has undergone somatic mutation can contain framework residues that differ from the germline sequence from which the antibody is derived. Such residues can be identified by comparing the antibody or fragment framework sequences to the germline sequences from which the antibody or fragment is derived. Another approach is to revert to the original parental (e.g., rodent) residue at one or more positions of the engineered (e.g. humanized) antibody, e.g. to restore binding affinity that may have been lost in the process of replacing the framework residues. (See, e.g., U.S. Pat. Nos. 5,693,762, 5,585,089 and 5,530,101.)

In certain embodiments, the anti-CD27 antibodies and antigen-binding fragments thereof are engineered (e.g. humanized) to include modifications in the framework and/or CDRs to improve their properties. Such engineered changes can be based on molecular modeling. A molecular model for the variable region for the parental (non-human) antibody sequence can be constructed to understand the structural features of the antibody and used to identify potential regions on the antibody that can interact with the antigen. Conventional CDRs are based on alignment of immunoglobulin sequences and identifying variable regions. Kabat et al., (1991) Sequences of Proteins of Immunological Interest, Kabat, et al.; National Institutes of Health, Bethesda, Md.; 5^(th) ed.; NIH Publ. No. 91-3242; Kabat (1978) Adv. Prot. Chem. 32:1-75; Kabat, et al., (1977) J. Biol. Chem. 252:6609-6616. Chothia and coworkers carefully examined conformations of the loops in crystal structures of antibodies and proposed hypervariable loops. Chothia, et al., (1987) J Mol. Biol. 196:901-917 or Chothia, et al., (1989) Nature 342:878-883. There are variations between regions classified as “CDRs” and “hypervariable loops”. Later studies (Raghunathan et al, (2012) J. Mol Recog. 25, 3, 103-113) analyzed several antibody-antigen crystal complexes and observed that the antigen binding regions in antibodies do not necessarily conform strictly to the “CDR” residues or “hypervariable” loops. The molecular model for the variable region of the non-human antibody can be used to guide the selection of regions that can potentially bind to the antigen. In practice, the potential antigen binding regions based on model differ from the conventional “CDR”s or “hyper variable” loops. Commercial scientific software such as MOE (Chemical Computing Group) can be used for molecular modeling. Human frameworks can be selected based on best matches with the non-human sequence both in the frameworks and in the CDRs. For FR4 (framework 4) in VH, VJ regions for the human germlines are compared with the corresponding non-human region. In the case of FR4 (framework 4) in VL, J-kappa and J-Lambda regions of human germline sequences are compared with the corresponding non-human region. Once suitable human frameworks are identified, the CDRs are grafted into the selected human frameworks. In some cases certain residues in the VL-VH interface can be retained as in the non-human (parental) sequence. Molecular models can also be used for identifying residues that can potentially alter the CDR conformations and hence binding to antigen. In some cases, these residues are retained as in the non-human (parental) sequence. Molecular models can also be used to identify solvent exposed amino acids that can result in unwanted effects such as glycosylation, deamidation and oxidation. Developability filters can be introduced early on in the design stage to eliminate/minimize these potential problems.

Another type of framework modification involves mutating one or more residues within the framework region, or even within one or more CDR regions, to remove T cell epitopes to thereby reduce the potential immunogenicity of the antibody. This approach is also referred to as “deimmunization” and is described in further detail in U.S. Pat. No. 7,125,689.

In particular embodiments, it will be desirable to change certain amino acids containing exposed side-chains to another amino acid residue in order to provide for greater chemical stability of the final antibody, so as to avoid deamidation or isomerization. The deamidation of asparagine may occur on NG, DG, NG, NS, NA, NT, QG or QS sequences and result in the creation of an isoaspartic acid residue that introduces a kink into the polypeptide chain and decreases its stability (isoaspartic acid effect). Isomerization can occur at DG, DS, DA or DT sequences. In certain embodiments, the antibodies of the present disclosure do not contain deamidation or asparagine isomerism sites.

For example, an asparagine (Asn) residue may be changed to Gln or Ala to reduce the potential for formation of isoaspartate at any Asn-Gly sequences, particularly within a CDR. A similar problem may occur at a Asp-Gly sequence. Reissner and Aswad (2003) Cell. Mol. Life Sci. 60:1281. Isoaspartate formation may debilitate or completely abrogate binding of an antibody to its target antigen. See, Presta (2005) J. Allergy Clin. Immunol. 116:731 at 734. In one embodiment, the asparagine is changed to glutamine (Gln). It may also be desirable to alter an amino acid adjacent to an asparagine (Asn) or glutamine (Gln) residue to reduce the likelihood of deamidation, which occurs at greater rates when small amino acids occur adjacent to asparagine or glutamine. See, Bischoff & Kolbe (1994) J. Chromatog. 662:261. In addition, any methionine residues (typically solvent exposed Met) in CDRs may be changed to Lys, Leu, Ala, or Phe or other amino acids in order to reduce the possibility that the methionine sulfur would oxidize, which could reduce antigen-binding affinity and also contribute to molecular heterogeneity in the final antibody preparation. Id. Additionally, in order to prevent or minimize potential scissile Asn-Pro peptide bonds, it may be desirable to alter any Asn-Pro combinations found in a CDR to Gln-Pro, Ala-Pro, or Asn-Ala. Antibodies with such substitutions are subsequently screened to ensure that the substitutions do not decrease the affinity or specificity of the antibody for CD27, or other desired biological activity to unacceptable levels.

TABLE 2 Exemplary stabilizing CDR variants CDR Residue Stabilizing Variant Sequence Asn-Gly Gln-Gly, Ala-Gly, or Asn-Ala (N-G) (Q-G), (A-G), or (N-A) Asp-Gly Glu-Gly, Ala-Gly or Asp-Ala (D-G) (E-G), (A-G), or (D-A) Met Lys, Leu, Ala, or Phe (M) (K), (L), (A), or (F) Asn Gln or Ala (N) (Q) or (A) Asn-Pro Gln-Pro, Ala-Pro, or Asn-Ala (N-P) (Q-P), (A-P), or (N-A)

Antibody Engineering of the Fc Region

The antibodies (e.g., humanized antibodies) and antigen-binding fragments thereof disclosed herein (e.g., antibody 131A and humanized versions thereof) can also be engineered to include modifications within the Fc region, typically to alter one or more properties of the antibody, such as serum half-life, complement fixation, Fc receptor binding, and/or effector function (e.g., antigen-dependent cellular cytotoxicity). Furthermore, the antibodies and antigen-binding fragments thereof disclosed herein (e.g., antibody 131A and humanized versions thereof) can be chemically modified (e.g., one or more chemical moieties can be attached to the antibody) or be modified to alter its glycosylation, again to alter one or more properties of the antibody or fragment. Each of these embodiments is described in further detail below. The numbering of residues in the Fc region is that of the EU index of Kabat.

The antibodies and antigen-binding fragments thereof disclosed herein (e.g., antibody 131A and humanized versions thereof) also include antibodies and fragments with modified (or blocked) Fc regions to provide altered effector functions. See, e.g., U.S. Pat. No. 5,624,821; WO2003/086310; WO2005/120571; WO2006/0057702. Such modifications can be used to enhance or suppress various reactions of the immune system, with possible beneficial effects in diagnosis and therapy. Alterations of the Fc region include amino acid changes (substitutions, deletions and insertions), glycosylation or deglycosylation, and adding multiple Fc regions. Changes to the Fc can also alter the half-life of antibodies in therapeutic antibodies, enabling less frequent dosing and thus increased convenience and decreased use of material. See Presta (2005) J. Allergy Clin. Immunol. 116:731 at 734-35.

In one embodiment, the antibody or antigen-binding fragment of the invention (e.g., antibody 131A and humanized versions thereof) is an IgG4 isotype antibody or fragment comprising a Serine to Proline mutation at a position corresponding to position 228 (S228P; EU index) in the hinge region of the heavy chain constant region. This mutation has been reported to abolish the heterogeneity of inter-heavy chain disulfide bridges in the hinge region (Angal et al. supra; position 241 is based on the Kabat numbering system).

In one embodiment of the invention, the hinge region of CH1 is modified such that the number of cysteine residues in the hinge region is increased or decreased. This approach is described further in U.S. Pat. No. 5,677,425. The number of cysteine residues in the hinge region of CH1 is altered, for example, to facilitate assembly of the light and heavy chains or to increase or decrease the stability of the antibody.

In another embodiment, the Fc hinge region of an antibody or antigen-binding fragment of the invention (e.g., antibody 131A and humanized versions thereof) is mutated to decrease the biological half-life of the antibody or fragment. More specifically, one or more amino acid mutations are introduced into the CH2-CH3 domain interface region of the Fc-hinge fragment such that the antibody or fragment has impaired Staphylococcyl protein A (SpA) binding relative to native Fc-hinge domain SpA binding. This approach is described in further detail in U.S. Pat. No. 6,165,745.

In another embodiment, the antibody or antigen-binding fragment of the invention (e.g., antibody 131A and humanized versions thereof) is modified to increase its biological half-life. Various approaches are possible. For example, one or more of the following mutations can be introduced: T252L, T254S, T256F, as described in U.S. Pat. No. 6,277,375. Alternatively, to increase the biological half-life, the antibody can be altered within the CH1 or CL region to contain a salvage receptor binding epitope taken from two loops of a CH2 domain of an Fc region of an IgG, as described in U.S. Pat. Nos. 5,869,046 and 6,121,022.

In yet other embodiments, the Fc region is altered by replacing at least one amino acid residue with a different amino acid residue to alter the effector function(s) of the antibody or antigen-binding fragment. For example, one or more amino acids selected from amino acid residues 234, 235, 236, 237, 297, 318, 320 and 322 can be replaced with a different amino acid residue such that the antibody has an altered affinity for an effector ligand and retains the antigen-binding ability of the parent antibody. The effector ligand to which affinity is altered can be, for example, an Fc receptor or the C1 component of complement. This approach is described in further detail in U.S. Pat. Nos. 5,624,821 and 5,648,260.

In another example, one or more amino acids selected from amino acid residues 329, 331 and 322 can be replaced with a different amino acid residue such that the antibody has altered C1q binding and/or reduced or abolished complement dependent cytotoxicity (CDC). This approach is described in further detail in U.S. Pat. No. 6,194,551.

In another example, one or more amino acid residues within amino acid positions 231 and 239 are altered to thereby alter the ability of the antibody to fix complement. This approach is described further in PCT Publication WO 94/29351.

In yet another example, the Fc region is modified to decrease the ability of the antibody or antigen-binding fragment of the invention (e.g., antibody 131A and humanized versions thereof) to mediate antibody dependent cellular cytotoxicity (ADCC) and/or to decrease the affinity of the antibody or fragment for an Fcγ receptor by modifying one or more amino acids at the following positions: 238, 239, 243, 248, 249, 252, 254, 255, 256, 258, 264, 265, 267, 268, 269, 270, 272, 276, 278, 280, 283, 285, 286, 289, 290, 292, 293, 294, 295, 296, 298, 301, 303, 305, 307, 309, 312, 315, 320, 322, 324, 326, 327, 329, 330, 331, 333, 334, 335, 337, 338, 340, 360, 373, 376, 378, 382, 388, 389, 398, 414, 416, 419, 430, 434, 435, 437, 438 or 439. This approach is described further in PCT Publication WO 00/42072. Moreover, the binding sites on human IgG1 for FcγR1, FcγRII, FcγRIII and FcRn have been mapped and variants with improved binding have been described (see Shields et al. (2001)J. Biol. Chem. 276:6591-6604).

In one embodiment of the invention, the Fc region is modified to decrease the ability of the antibody of the invention (e.g., antibody 131A and humanized versions thereof) to mediate effector function and/or to increase anti-inflammatory properties by modifying residues 243 and 264. In one embodiment, the Fc region of the antibody or fragment is modified by changing the residues at positions 243 and 264 to alanine. In one embodiment, the Fc region is modified to decrease the ability of the antibody or fragment to mediate effector function and/or to increase anti-inflammatory properties by modifying residues 243, 264, 267 and 328.

Altered Effector Function

In some embodiments, the Fc region of an anti-CD27 antibody is modified to increase or reduce the ability of the antibody or antigen-binding fragment to mediate effector function and/or to increase/decrease their binding to the Fcgamma receptors (FcγRs).

The term “Effector Function” as used herein is meant to refer to one or more of Antibody Dependant Cell mediated Cytotoxic activity (ADCC), Complement-dependant cytotoxic activity (CDC) mediated responses, Fc-mediated phagocytosis or antibody dependant cellular phagocytosis (ADCP) and antibody recycling via the FcRn receptor.

The interaction between the constant region of an antigen binding protein and various Fc receptors (FcR) including FcgammaRI (CD64), FcgammaRII (CD32) and FcgammaRIII (CD16) is believed to mediate the effector functions, such as ADCC and CDC, of the antigen binding protein. The Fc receptor is also important for antibody cross-linking, which can be important for anti-tumor immunity.

Effector function can be measured in a number of ways including for example via binding of the FcgammaRIII to Natural Killer cells or via FcgammaRI to monocytes/macrophages to measure for ADCC effector function. For example an antigen binding protein of the present invention can be assessed for ADCC effector function in a Natural Killer cell assay. Examples of such assays can be found in Shields et al, 2001 J. Biol. Chem., Vol. 276, p 6591-6604; Chappel et al, 1993 J. Biol. Chem., Vol 268, p 25124-25131; Lazar et al, 2006 PNAS, 103; 4005-4010.

Human IgG1 constant regions containing specific mutations or altered glycosylation on residue Asn297 have been shown to reduce binding to Fc receptors. In other cases, mutations have also been shown to enhance ADCC and CDC (Lazar et al. PNAS 2006, 103; 4005-4010; Shields et al. J Biol Chem 2001, 276; 6591-6604; Nechansky et al. Mol Immunol, 2007, 44; 1815-1817).

In one embodiment of the present invention, such mutations are in one or more of positions selected from 239, 332 and 330 (IgG1), or the equivalent positions in other IgG isotypes. Examples of suitable mutations are S239D and I332E and A330L. In one embodiment, the antigen binding protein of the invention herein described is mutated at positions 239 and 332, for example S239D and I332E or in a further embodiment it is mutated at three or more positions selected from 239 and 332 and 330, for example S239D and I332E and A330L. (EU index numbering).

In an alternative embodiment of the present invention, there is provided an antibody comprising a heavy chain constant region with an altered glycosylation profile such that the antigen binding protein has enhanced effector function. For example, wherein the antibody has enhanced ADCC or enhanced CDC or wherein it has both enhanced ADCC and CDC effector function. Examples of suitable methodologies to produce antigen binding proteins with an altered glycosylation profile are described in WO2003011878, WO2006014679 and EP1229125.

In a further aspect, the present invention provides “non-fucosylated” or “afucosylated” antibodies. Non-fucosylated antibodies harbor a tri-mannosyl core structure of complex-type N-glycans of Fc without fucose residue. These glycoengineered antibodies that lack core fucose residue from the Fc N-glycans may exhibit stronger ADCC than fucosylated equivalents due to enhancement of FcgammaRIIIa binding capacity.

The present invention also provides a method for the production of an antibody according to the invention comprising the steps of: a) culturing a recombinant host cell comprising an expression vector comprising the isolated nucleic acid as described herein, wherein the recombinant host cell does not comprise an alpha-1,6-fucosyltransferase; and b) recovering the antigen binding protein. The recombinant host cell may not normally contain a gene encoding an alpha-1,6-fucosyltransferase (for example yeast host cells such as Pichia sp.) or may have been genetically modified to inactivate an alpha-1,6-fucosyltransferase. Recombinant host cells which have been genetically modified to inactivate the FUT8 gene encoding an alpha-1,6-fucosyltransferase are available. See, e.g., the POTELLIGENT™ technology system available from BioWa, Inc. (Princeton, N.J.) in which CHOK1SV cells lacking a functional copy of the FUT8 gene produce monoclonal antibodies having enhanced antibody dependent cell mediated cytotoxicity (ADCC) activity that is increased relative to an identical monoclonal antibody produced in a cell with a functional FUT8 gene. Aspects of the POTELLIGENT™ technology system are described in U.S. Pat. Nos. 7,214,775, 6,946,292, WO0061739 and WO0231240. Those of ordinary skill in the art will also recognize other appropriate systems.

It will be apparent to those skilled in the art that such modifications may not only be used alone but may be used in combination with each other in order to further enhance or decrease effector function.

Production of Antibodies with Modified Glycosylation

In still another embodiment, the antibodies or antigen-binding fragments of the invention (e.g., antibody 131A and humanized versions thereof) comprise a particular glycosylation pattern. For example, an afucosylated or an aglycosylated antibody or fragment can be made (i.e., the antibody lacks fucose or glycosylation, respectively). The glycosylation pattern of an antibody or fragment may be altered to, for example, increase the affinity or avidity of the antibody or fragment for a CD27 antigen. Such modifications can be accomplished by, for example, altering one or more of the glycosylation sites within the antibody or fragment sequence. For example, one or more amino acid substitutions can be made that result in removal of one or more of the variable region framework glycosylation sites to thereby eliminate glycosylation at that site. Such aglycosylation may increase the affinity or avidity of the antibody or fragment for antigen. See, e.g., U.S. Pat. Nos. 5,714,350 and 6,350,861.

Antibodies and antigen-binding fragments disclosed herein (e.g., antibody 131A and humanized versions thereof) may further include those produced in lower eukaryote host cells, in particular fungal host cells such as yeast and filamentous fungi have been genetically engineered to produce glycoproteins that have mammalian- or human-like glycosylation patterns (See for example, Choi et al, (2003) Proc. Natl. Acad. Sci. 100: 5022-5027; Hamilton et al., (2003) Science 301: 1244-1246; Hamilton et al., (2006) Science 313: 1441-1443; Nett et al., Yeast 28(3):237-52 (2011); Hamilton et al., Curr Opin Biotechnol. October; 18(5):387-92 (2007)). A particular advantage of these genetically modified host cells over currently used mammalian cell lines is the ability to control the glycosylation profile of glycoproteins that are produced in the cells such that compositions of glycoproteins can be produced wherein a particular N-glycan structure predominates (see, e.g., U.S. Pat. Nos. 7,029,872 and 7,449,308). These genetically modified host cells have been used to produce antibodies that have predominantly particular N-glycan structures (See for example, Li et al., (2006) Nat. Biotechnol. 24: 210-215).

In particular embodiments, the antibodies and antigen-binding fragments thereof disclosed herein (e.g., antibody 131A and humanized versions thereof) further include those produced in lower eukaryotic host cells and which comprise fucosylated and non-fucosylated hybrid and complex N-glycans, including bisected and multiantennary species, including but not limited to N-glycans such as GlcNAc₍₁₋₄₎Man₃GlcNAc₂; Gal₍₁₋₄₎GlcNAc₍₁₋₄₎Man₃GlcNAc₂; NANA₍₁₋₄₎Gal₍₁₋₄₎GlcNAc₍₁₋₄₎Man₃GlcNAc₂.

In particular embodiments, the antibodies and antigen-binding fragments thereof provided herein (e.g., antibody 131A and humanized versions thereof) may comprise antibodies or fragments having at least one hybrid N-glycan selected from the group consisting of GlcNAcMan₅GlcNAc₂; GalGlcNAcMan₅GlcNAc₂; and NANAGalGlcNAcMan₅GlcNAc₂. In particular aspects, the hybrid N-glycan is the predominant N-glycan species in the composition.

In particular embodiments, the antibodies and antigen-binding fragments thereof provided herein (e.g., antibody 131A and humanized versions thereof) comprise antibodies and fragments having at least one complex N-glycan selected from the group consisting of GlcNAcMan₃GlcNAc₂; GalGlcNAcMan₃GlcNAc₂; NANAGalGlcNAcMan₃GlcNAc₂; GlcNAc₂Man₃GlcNAc₂; GalGlcNAc₂Man₃GlcNAc₂; Gal₂GlcNAc₂Man₃GlcNAc₂; NANAGal₂GlcNAc₂Man₃GlcNAc₂; and NANA₂Gal₂GlcNAc₂Man₃GlcNAc₂. In particular aspects, the complex N-glycan are the predominant N-glycan species in the composition. In further aspects, the complex N-glycan is a particular N-glycan species that comprises about 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 97%, 98%, 99%, or 100% of the complex N-glycans in the composition. In one embodiment, the antibody and antigen binding fragments thereof provided herein comprise complex N-glycans, wherein at least 50%, 60%, 70%, 80%, 90%, 95%, 97%, 98%, 99%, or 100% of the complex N-glycans comprise the structure NANA₂Gal₂GlcNAc₂Man₃GlcNAc₂, wherein such structure is afucosylated. Such structures can be produced, e.g., in engineered Pichia pastoris host cells.

In particular embodiments, the N-glycan is fucosylated. In general, the fucose is in an α1,3-linkage with the GlcNAc at the reducing end of the N-glycan, an α1,6-linkage with the GlcNAc at the reducing end of the N-glycan, an α1,2-linkage with the Gal at the non-reducing end of the N-glycan, an α1,3-linkage with the GlcNac at the non-reducing end of the N-glycan, or an α1,4-linkage with a GlcNAc at the non-reducing end of the N-glycan.

Therefore, in particular aspects of the above the glycoprotein compositions, the glycoform is in an α1,3-linkage or α1,6-linkage fucose to produce a glycoform selected from the group consisting of Man₅GlcNAc₂(Fuc), GlcNAcMan₅GlcNAc₂(Fuc), Man₃GlcNAc₂(Fuc), GlcNAcMan₃GlcNAc₂(Fuc), GlcNAc₂Man₃GlcNAc₂(Fuc), GalGlcNAc₂Man₃GlcNAc₂(Fuc), Gal₂GlcNAc₂Man₃GlcNAc₂(Fuc), NANAGal₂GlcNAc₂Man₃GlcNAc₂(Fuc), and NANA₂Gal₂GlcNAc₂Man₃GlcNAc₂(Fuc); in an α1,3-linkage or α1,4-linkage fucose to produce a glycoform selected from the group consisting of GlcNAc(Fuc)Man₅GlcNAc₂, GlcNAc(Fuc)Man₃GlcNAc₂, GlcNAc₂(Fuc₁₋₂)Man₃GlcNAc₂, GalGlcNAc₂(Fuc₁₋₂)Man₃GlcNAc₂, Gal₂GlcNAc₂(Fuc₁₋₂)Man₃GlcNAc₂, NANAGal₂GlcNAc₂(Fuc₁₋₂)Man₃GlcNAc₂, and NANA₂Gal₂GlcNAc₂(Fuc₁₋₂)Man₃GlcNAc₂; or in an α1,2-linkage fucose to produce a glycoform selected from the group consisting of Gal(Fuc)GlcNAc₂Man₃GlcNAc₂, Gal₂(Fuc₁₋₂)GlcNAc₂Man₃GlcNAc₂, NANAGal₂(Fuc₁₋₂)GlcNAc₂Man₃GlcNAc₂, and NANA₂Gal₂(Fuc₁₋₂)GlcNAc₂Man₃GlcNAc₂.

In further aspects, the antibodies (e.g., humanized antibodies) or antigen-binding fragments thereof comprise high mannose N-glycans, including but not limited to, Man₅GlcNAc₂, Man₇GlcNAc₂, Man₆GlcNAc₂, Man₅GlcNAc₂, Man₄GlcNAc₂, or N-glycans that consist of the Man₃GlcNAc₂ N-glycan structure.

In further aspects of the above, the complex N-glycans further include fucosylated and non-fucosylated bisected and multiantennary species.

As used herein, the terms “N-glycan” and “glycoform” are used interchangeably and refer to an N-linked oligosaccharide, for example, one that is attached by an asparagine-N-acetylglucosamine linkage to an asparagine residue of a polypeptide. N-linked glycoproteins contain an N-acetylglucosamine residue linked to the amide nitrogen of an asparagine residue in the protein. The predominant sugars found on glycoproteins are glucose, galactose, mannose, fucose, N-acetylgalactosamine (GalNAc), N-acetylglucosamine (GlcNAc) and sialic acid (e.g., N-acetyl-neuraminic acid (NANA)). The processing of the sugar groups occurs co-translationally in the lumen of the ER and continues post-translationally in the Golgi apparatus for N-linked glycoproteins.

N-glycans have a common pentasaccharide core of Man₃GlcNAc₂ (“Man” refers to mannose; “Glc” refers to glucose; and “NAc” refers to N-acetyl; GlcNAc refers to N-acetylglucosamine). Usually, N-glycan structures are presented with the non-reducing end to the left and the reducing end to the right. The reducing end of the N-glycan is the end that is attached to the Asn residue comprising the glycosylation site on the protein. N-glycans differ with respect to the number of branches (antennae) comprising peripheral sugars (e.g., GlcNAc, galactose, fucose and sialic acid) that are added to the Man₃GlcNAc₂ (“Man₃”) core structure which is also referred to as the “trimannose core”, the “pentasaccharide core” or the “paucimannose core”. N-glycans are classified according to their branched constituents (e.g., high mannose, complex or hybrid). A “high mannose” type N-glycan has five or more mannose residues. A “complex” type N-glycan typically has at least one GlcNAc attached to the 1,3 mannose arm and at least one GlcNAc attached to the 1,6 mannose arm of a “trimannose” core. Complex N-glycans may also have galactose (“Gal”) or N-acetylgalactosamine (“GalNAc”) residues that are optionally modified with sialic acid or derivatives (e.g., “NANA” or “NeuAc”, where “Neu” refers to neuraminic acid and “Ac” refers to acetyl). Complex N-glycans may also have intrachain substitutions comprising “bisecting” GlcNAc and core fucose (“Fuc”). Complex N-glycans may also have multiple antennae on the “trimannose core,” often referred to as “multiple antennary glycans.” A “hybrid” N-glycan has at least one GlcNAc on the terminal of the 1,3 mannose arm of the trimannose core and zero or more mannoses on the 1,6 mannose arm of the trimannose core. The various N-glycans are also referred to as “glycoforms”.

With respect to complex N-glycans, the terms “G-2”, “G-1”, “G0”, “G1”, “G2”, “A1”, and “A2” mean the following. “G-2” refers to an N-glycan structure that can be characterized as Man₃GlcNAc₂; the term “G-1” refers to an N-glycan structure that can be characterized as GlcNAcMan₃GlcNAc₂; the term “G0” refers to an N-glycan structure that can be characterized as GlcNAc₂Man₃GlcNAc₂; the term “G1” refers to an N-glycan structure that can be characterized as GalGlcNAc₂Man₃GlcNAc₂; the term “G2” refers to an N-glycan structure that can be characterized as Gal₂GlcNAc₂Man₃GlcNAc₂; the term “A1” refers to an N-glycan structure that can be characterized as NANAGal₂GlcNAc₂Man₃GlcNAc₂; and, the term “A2” refers to an N-glycan structure that can be characterized as NANA₂Gal₂GlcNAc₂Man₃GlcNAc₂. Unless otherwise indicated, the terms G-2”, “G-1”, “G0”, “G1”, “G2”, “A1”, and “A2” refer to N-glycan species that lack fucose attached to the GlcNAc residue at the reducing end of the N-glycan. When the term includes an “F”, the “F” indicates that the N-glycan species contains a fucose residue on the GlcNAc residue at the reducing end of the N-glycan. For example, G0F, G1F, G2F, A1F, and A2F all indicate that the N-glycan further includes a fucose residue attached to the GlcNAc residue at the reducing end of the N-glycan. Lower eukaryotes such as yeast and filamentous fungi do not normally produce N-glycans that produce fucose.

With respect to multiantennary N-glycans, the term “multiantennary N-glycan” refers to N-glycans that further comprise a GlcNAc residue on the mannose residue comprising the non-reducing end of the 1,6 arm or the 1,3 arm of the N-glycan or a GlcNAc residue on each of the mannose residues comprising the non-reducing end of the 1,6 arm and the 1,3 arm of the N-glycan. Thus, multiantennary N-glycans can be characterized by the formulas GlcNAc₍₂₋₄₎Man₃GlcNAc₂, Gal₍₁₋₄₎GlcNAc₍₂₋₄₎Man₃GlcNAc₂, or NANA₍₁₋₄₎Gal₍₁₋₄₎GlcNAc₍₂₋₄₎Man₃GlcNAc₂. The term “1-4” refers to 1, 2, 3, or 4 residues.

With respect to bisected N-glycans, the term “bisected N-glycan” refers to N-glycans in which a GlcNAc residue is linked to the mannose residue at the reducing end of the N-glycan. A bisected N-glycan can be characterized by the formula GlcNAc₃Man₃GlcNAc₂ wherein each mannose residue is linked at its non-reducing end to a GlcNAc residue. In contrast, when a multiantennary N-glycan is characterized as GlcNAc₃Man₃GlcNAc₂, the formula indicates that two GlcNAc residues are linked to the mannose residue at the non-reducing end of one of the two arms of the N-glycans and one GlcNAc residue is linked to the mannose residue at the non-reducing end of the other arm of the N-glycan.

Antibody Physical Properties

The antibodies and antigen-binding fragments thereof disclosed herein (e.g., antibody 131A and humanized versions thereof) may further contain one or more glycosylation sites in either the light or heavy chain immunoglobulin variable region. Such glycosylation sites may result in increased immunogenicity of the antibody or fragment or an alteration of the pK of the antibody due to altered antigen-binding (Marshall et al. (1972) Annu Rev Biochem 41:673-702; Gala and Morrison (2004) J Immunol 172:5489-94; Wallick et al (1988) JExp Med 168:1099-109; Spiro (2002) Glycobiology 12:43R-56R; Parekh et al (1985) Nature 316:452-7; Mimura et al. (2000) Mol Immunol 37:697-706). Glycosylation has been known to occur at motifs containing an N—X-S/T sequence.

Each antibody or antigen-binding fragment (e.g., 131A or humanized versions thereof) will have a unique isoelectric point (pI), which generally falls in the pH range between 6 and 9.5. The pI for an IgG1 antibody typically falls within the pH range of 7-9.5 and the pI for an IgG4 antibody typically falls within the pH range of 6-8.

Each antibody or antigen-binding fragment (e.g., 131A or humanized versions thereof) will have a characteristic melting temperature, with a higher melting temperature indicating greater overall stability in vivo (Krishnamurthy R and Manning M C (2002) Curr Pharm Biotechnol 3:361-71). In general, the T_(M1) (the temperature of initial unfolding) may be greater than 60° C., greater than 65° C., or greater than 70° C. The melting point of an antibody or fragment can be measured using differential scanning calorimetry (Chen et al (2003) Pharm Res 20:1952-60; Ghirlando et al (1999) Immunol Lett 68:47-52) or circular dichroism (Murray et al. (2002) J Chromatogr Sci 40:343-9).

In a further embodiment, antibodies and antigen-binding fragments thereof (e.g., antibody 131A and humanized versions thereof) are selected that do not degrade rapidly. Degradation of an antibody or fragment can be measured using capillary electrophoresis (CE) and MALDI-MS (Alexander A J and Hughes D E (1995) Anal Chem 67:3626-32).

In a further embodiment, antibodies (e.g., antibody 131A and humanized versions thereof) and antigen-binding fragments thereof are selected that have minimal aggregation effects, which can lead to the triggering of an unwanted immune response and/or altered or unfavorable pharmacokinetic properties. Generally, antibodies and fragments are acceptable with aggregation of 25% or less, 20% or less, 15% or less, 10% or less, or 5% or less. Aggregation can be measured by several techniques, including size-exclusion column (SEC), high performance liquid chromatography (HPLC), and light scattering.

Antibody Conjugates

The anti-CD27 antibodies and antigen-binding fragments thereof disclosed herein (e.g., antibody 131A and humanized versions thereof) may also be conjugated to a chemical moiety. The chemical moiety may be, inter alia, a polymer, a radionucleotide or a cytotoxic factor. In particular embodiments, the chemical moiety is a polymer which increases the half-life of the antibody or fragment in the body of a subject. Suitable polymers include, but are not limited to, hydrophilic polymers which include but are not limited to polyethylene glycol (PEG) (e.g., PEG with a molecular weight of 2 kDa, 5 kDa, 10 kDa, 12 kDa, 20 kDa, 30 kDa or 40 kDa), dextran and monomethoxypolyethylene glycol (mPEG). Lee, et al., (1999) (Bioconj. Chem. 10:973-981) discloses PEG conjugated single-chain antibodies. Wen, et al., (2001) (Bioconj. Chem. 12:545-553) disclose conjugating antibodies with PEG which is attached to a radiometal chelator (diethylenetriaminpentaacetic acid (DTPA)).

The antibodies and antigen-binding fragments thereof disclosed herein (e.g., antibody 131A and humanized versions thereof) may also be conjugated with labels such as ⁹⁹Tc, ⁹⁰Y, ¹¹¹In, ³²P, ¹⁴C, ¹²⁵I, ³H, ¹³¹I, C, ¹⁵O, ¹³N, ¹⁸F, ³⁵S, ⁵¹Cr, ⁵⁷To, ²²⁶Ra, ⁶⁰Co, ⁵⁹Fe, ⁵⁷Se, ¹⁵²Eu, ⁶⁷CU, ²¹⁷Ci, ²¹¹At, ²¹²Pb, ⁴⁷Sc, ¹⁰⁹Pd, ²³⁴Th, and ⁴⁰K, ¹⁵⁷Gd, ⁵⁵Mn, ⁵²Tr, and ⁵⁶Fe.

The antibodies and antigen-binding fragments disclosed herein (e.g., antibody 131A and humanized versions thereof) may also be PEGylated, for example to increase its biological (e.g., serum) half-life. To PEGylate an antibody or fragment, the antibody or fragment, typically is reacted with a reactive form of polyethylene glycol (PEG), such as a reactive ester or aldehyde derivative of PEG, under conditions in which one or more PEG groups become attached to the antibody or antibody fragment. In particular embodiments, the PEGylation is carried out via an acylation reaction or an alkylation reaction with a reactive PEG molecule (or an analogous reactive water-soluble polymer). As used herein, the term “polyethylene glycol” is intended to encompass any of the forms of PEG that have been used to derivatize other proteins, such as mono (C1-C10) alkoxy- or aryloxy-polyethylene glycol or polyethylene glycol-maleimide. In certain embodiments, the antibody or fragment to be PEGylated is an aglycosylated antibody or fragment. Methods for PEGylating proteins are known in the art and can be applied to the antibodies of the invention. See, e.g., EP 0 154 316 and EP 0 401 384.

The antibodies and antigen-binding fragments disclosed herein (e.g., antibody 131A and humanized versions thereof) may also be conjugated with fluorescent or chemilluminescent labels, including fluorophores such as rare earth chelates, fluorescein and its derivatives, rhodamine and its derivatives, isothiocyanate, phycoerythrin, phycocyanin, allophycocyanin, o-phthaladehyde, fluorescamine, ¹⁵²Eu, dansyl, umbelliferone, luciferin, luminal label, isoluminal label, an aromatic acridinium ester label, an imidazole label, an acridimium salt label, an oxalate ester label, an aequorin label, 2,3-dihydrophthalazinediones, biotin/avidin, spin labels and stable free radicals.

The antibodies and antigen-binding fragments thereof of the invention (e.g., antibody 131A and humanized versions thereof) may also be conjugated to a cytotoxic factor such as diptheria toxin, Pseudomonas aeruginosa exotoxin A chain, ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins and compounds (e.g., fatty acids), dianthin proteins, Phytoiacca americana proteins PAPI, PAPII, and PAP-S, Momordica charantia inhibitor, curcin, crotin, Saponaria officinalis inhibitor, mitogellin, restrictocin, phenomycin, and enomycin.

Any method known in the art for conjugating the antibodies and antigen-binding fragments thereof of the invention (e.g., antibody 131A and humanized versions thereof) to the various moieties may be employed, including those methods described by Hunter, et al., (1962) Nature 144:945; David, et al., (1974) Biochemistry 13:1014; Pain, et al., (1981) J. Immunol. Meth. 40:219; and Nygren, J., (1982) Histochem. and Cytochem. 30:407. Methods for conjugating antibodies and fragments are conventional and very well known in the art.

Therapeutic Uses of Anti-CD27 Antibodies

Further provided are methods for treating subjects, including human subjects, in need of treatment with the isolated antibodies or antigen-binding fragments thereof disclosed herein (e.g., antibody 131A and humanized versions thereof). In one embodiment of the invention, such subject suffers from an infection or an infectious disease. The invention also provides an antibody or antigen binding fragment of the invention for use in treatment of cancer; or treatment of an infection or infectious disease. The invention also provides the use of the antibody or antigen binding fragment of the invention for the manufacture of a medicament for increasing immune cell activation; treating cancer; or treating an infection or infectious disease.

In another embodiment of the invention, such subject suffers from cancer. In one embodiment the cancer is, e.g., osteosarcoma, rhabdomyosarcoma, neuroblastoma, kidney cancer, leukemia, renal transitional cell cancer, bladder cancer, Wilm's cancer, ovarian cancer, pancreatic cancer, breast cancer, prostate cancer, bone cancer, lung cancer (e.g., non-small cell lung cancer), gastric cancer, colorectal cancer, cervical cancer, synovial sarcoma, head and neck cancer, squamous cell carcinoma, multiple myeloma, renal cell cancer, retinoblastoma, hepatoblastoma, hepatocellular carcinoma, melanoma, rhabdoid tumor of the kidney, Ewing's sarcoma, chondrosarcoma, brain cancer, glioblastoma, meningioma, pituitary adenoma, vestibular schwannoma, a primitive neuroectodermal tumor, medulloblastoma, astrocytoma, anaplastic astrocytoma, oligodendroglioma, ependymoma, choroid plexus papilloma, polycythemia vera, thrombocythemia, idiopathic myelfibrosis, soft tissue sarcoma, thyroid cancer, endometrial cancer, carcinoid cancer or liver cancer, breast cancer or gastric cancer. In an embodiment of the invention, the cancer is metastatic cancer, e.g., of the varieties described above.

Cancers that may be treated by the antibodies or antigen-binding fragments, compositions and methods of the invention include, but are not limited to: Cardiac: sarcoma (angiosarcoma, fibrosarcoma, rhabdomyosarcoma, liposarcoma), myxoma, rhabdomyoma, fibroma, lipoma and teratoma; Lung: bronchogenic carcinoma (squamous cell, undifferentiated small cell, undifferentiated large cell, adenocarcinoma), alveolar (bronchiolar) carcinoma, bronchial adenoma, sarcoma, lymphoma, chondromatous hamartoma, mesothelioma; Gastrointestinal: esophagus (squamous cell carcinoma, adenocarcinoma, leiomyosarcoma, lymphoma), stomach (carcinoma, lymphoma, leiomyosarcoma), pancreas (ductal adenocarcinoma, insulinoma, glucagonoma, gastrinoma, carcinoid tumors, vipoma), small bowel (adenocarcinoma, lymphoma, carcinoid tumors, Karposi's sarcoma, leiomyoma, hemangioma, lipoma, neurofibroma, fibroma), large bowel (adenocarcinoma, tubular adenoma, villous adenoma, hamartoma, leiomyoma) colorectal; Genitourinary tract: kidney (adenocarcinoma, Wilm's tumor [nephroblastoma], lymphoma, leukemia), bladder and urethra (squamous cell carcinoma, transitional cell carcinoma, adenocarcinoma), prostate (adenocarcinoma, sarcoma), testis (seminoma, teratoma, embryonal carcinoma, teratocarcinoma, choriocarcinoma, sarcoma, interstitial cell carcinoma, fibroma, fibroadenoma, adenomatoid tumors, lipoma); Liver: hepatoma (hepatocellular carcinoma), cholangiocarcinoma, hepatoblastoma, angiosarcoma, hepatocellular adenoma, hemangioma; Bone: osteogenic sarcoma (osteosarcoma), fibrosarcoma, malignant fibrous histiocytoma, chondrosarcoma, Ewing's sarcoma, malignant lymphoma (reticulum cell sarcoma), multiple myeloma, malignant giant cell tumor chordoma, osteochronfroma (osteocartilaginous exostoses), benign chondroma, chondroblastoma, chondromyxofibroma, osteoid osteoma and giant cell tumors; Nervous system: skull (osteoma, hemangioma, granuloma, xanthoma, osteitis deformans), meninges (meningioma, meningiosarcoma, gliomatosis), brain (astrocytoma, medulloblastoma, glioma, ependymoma, germinoma [pinealoma], glioblastoma multiform, oligodendroglioma, schwannoma, retinoblastoma, congenital tumors), spinal cord neurofibroma, meningioma, glioma, sarcoma); Gynecological: uterus (endometrial carcinoma), cervix (cervical carcinoma, pre tumor cervical dysplasia), ovaries (ovarian carcinoma [serous cystadenocarcinoma, mucinous cystadenocarcinoma, unclassified carcinoma], granulosa thecal cell tumors, Sertoli-Leydig cell tumors, dysgerminoma, malignant teratoma), vulva (squamous cell carcinoma, intraepithelial carcinoma, adenocarcinoma, fibrosarcoma, melanoma), vagina (clear cell carcinoma, squamous cell carcinoma, botryoid sarcoma (embryonal rhabdomyosarcoma), fallopian tubes (carcinoma), breast; Hematologic: blood (myeloid leukemia [acute and chronic], acute lymphoblastic leukemia, chronic lymphocytic leukemia, myeloproliferative diseases, multiple myeloma, myelodysplastic syndrome), Hodgkin's disease, non-Hodgkin's lymphoma [malignant lymphoma]; Skin: malignant melanoma, basal cell carcinoma, squamous cell carcinoma, Karposi's sarcoma, moles dysplastic nevi, lipoma, angioma, dermatofibroma, keloids, psoriasis; and Adrenal glands: neuroblastoma. Thus, the term “cancerous cell” as provided herein, includes a cell afflicted by any one of the above-identified conditions.

In one embodiment, cancers that may be treated by the antibodies or antigen-binding fragments thereof disclosed herein, compositions and methods of the invention include, but are not limited to: lung cancer, pancreatic cancer, colon cancer, colorectal cancer, myeloid leukemia, acute myelogenous leukemia, chronic myelogenous leukemia, chronic myelomonocytic leukemia, thyroid cancer, myelodysplastic syndrome, bladder carcinoma, epidermal carcinoma, melanoma, breast cancer, prostate cancer, head and neck cancers, ovarian cancer, brain cancers, cancers of mesenchymal origin, sarcomas, tetracarcinomas, neuroblastomas, kidney carcinomas, hepatomas, non-Hodgkin's lymphoma, multiple myeloma, and anaplastic thyroid carcinoma.

In an embodiment, the invention provides methods for treating subjects using an anti-CD27 antibody or antigen-binding fragment thereof of the invention (e.g., antibody 131A and humanized versions thereof), wherein the subject suffers from a viral infection. In one embodiment, the viral infection is an infection with a virus selected from the group consisting of human immunodeficiency virus (HIV), hepatitis virus (A, B, or C), herpes virus (e.g., VZV, HSV-I, HAV-6, HSV-II, and CMV, Epstein Barr virus), adenovirus, influenza virus, flaviviruses, echovirus, rhinovirus, coxsackie virus, coronavirus, respiratory syncytial virus, mumps virus, rotavirus, measles virus, rubella virus, parvovirus, vaccinia virus, HTLV virus, dengue virus, papillomavirus, molluscum virus, poliovirus, rabies virus, JC virus or arboviral encephalitis virus.

In an embodiment, the invention provides methods for treating subjects using an anti-CD27 antibody or antigen-binding fragment thereof of the invention, wherein the subject suffers from a bacterial infection. In one embodiment, the bacterial infection is infection with a bacteria selected from the group consisting of Chlamydia, rickettsial bacteria, mycobacteria, staphylococci, streptococci, pneumonococci, meningococci and gonococci, klebsiella, proteus, serratia, pseudomonas, Legionella, Corynebacterium diphtheriae, Salmonella, bacilli, Vibrio cholerae, Clostridium tetan, Clostridium botulinum, Bacillus anthricis, Yersinia pestis, Mycobacterium leprae, Mycobacterium lepromatosis, and Borriella.

In an embodiment, the invention provides methods for treating subjects using an anti-CD27 antibody or antigen-binding fragment thereof of the invention, wherein the subject suffers from a fungal infection. In one embodiment, the fungal infection is an infection with a fungus selected from the group consisting of Candida (albicans, krusei, glabrata, tropicalis, etc.), Cryptococcus neoformans, Aspergillus (fumigatus, niger, etc.), Genus Mucorales (mucor, absidia, rhizopus), Sporothrix schenkii, Blastomyces dermatitidis, Paracoccidioides brasiliensis, Coccidioides immitis and Histoplasma capsulatum.

In an embodiment, the invention provides methods for treating subjects using an anti-CD27 antibody or antigen-binding fragment thereof of the invention, wherein the subject suffers from a parasitic infection. In one embodiment, the parasitic infection is an infection with a parasite selected from the group consisting of Entamoeba histolytica, Balantidium coli, Naegleria fowleri, Acanthamoeba, Giardia lambia, Cryptosporidium, Pneumocystis carinii, Plasmodium vivax, Babesia microti, Trypanosoma brucei, Trypanosoma cruzi, Leishmania donovani, Toxoplasma gondii and Nippostrongylus brasiliensis.

A “subject” may be a mammal such as a human, dog, cat, horse, cow, mouse, rat, monkey (e.g., cynomolgus monkey, e.g., Macaca fascicularis) or rabbit. In preferred embodiments of the invention, the subject is a human subject.

The term “in association with” indicates that the components administered in a method of the present invention (e.g., an anti-CD27 antibody (e.g., humanized antibody) or antigen-binding fragment thereof (e.g., antibody 131A or a humanized version thereof) along with an anti-cancer agent can be formulated into a single composition for simultaneous delivery or formulated separately into two or more compositions (e.g., a kit). Each component can be administered to a subject at a different time than when the other component is administered; for example, each administration may be given non-simultaneously (e.g., separately or sequentially) at several intervals over a given period of time. Moreover, the separate components may be administered to a subject by the same or by a different route.

In particular embodiments, the antibodies or antigen-binding fragments thereof disclosed herein (e.g., antibody 131A or humanized versions thereof) may be used alone, or in association with other, further therapeutic agents and/or therapeutic procedures, for treating or preventing any disease such as cancer, e.g., as discussed herein, in a subject in need of such treatment or prevention. Compositions, e.g., pharmaceutical compositions comprising a pharmaceutically acceptable carrier, comprising such antibodies and fragments in association with further therapeutic agents are also part of the present invention.

Therefore, the present invention provides a method of treating cancer in a human subject, comprising administering to the subject an effective amount of the antibody or antigen binding fragment disclosed herein, optionally in association with a further therapeutic agent or therapeutic procedure. The present invention also provides a method of treating an infection or infectious disease in a human subject, comprising administering to the subject an effective amount of the antibody or antigen binding fragment disclosed herein, optionally in association with a further therapeutic agent or therapeutic procedure. The present invention also provides a method of increasing the activity of an immune cell, comprising administering to a subject in need thereof an effective amount of an antibody or antigen binding fragment disclosed herein. In one embodiment, the method is used for: the treatment of cancer; the treatment of an infection or infectious disease; or as a vaccine adjuvant. In another embodiment, the present invention provides an antibody or antigen binding fragment of the invention, for use in: treatment of cancer; increasing the activity of an immune cell; or treatment of an infection or infectious disease in combination with a further therapeutic agent. In a further embodiment, the present invention provides use of the antibody or antigen binding fragment of the invention for the manufacture of a medicament for increasing immune cell activation; treating cancer; or treating an infection or infectious disease in combination with a further therapeutic agent. In another embodiment, the present invention provides a combination of an antibody or antigen binding fragment of the invention and a further therapeutic agent for the treatment of cancer; increasing the activity of an immune cell; or treatment of an infection or infectious disease.

In other embodiments, the invention provides a method of treating cancer or treating an infection or infectious disease in a human subject, comprising administering to the subject an effective amount of an antibody or antigen binding fragment of the invention, or an expression vector or a host cell according to the invention optionally in association with a further therapeutic agent or therapeutic procedure.

In particular embodiments, the antibodies or antigen-binding fragments thereof disclosed herein (e.g., antibody 131A and humanized versions thereof) may be used alone, or in association with tumor vaccines. Examples of tumor vaccines include but are not limited to vaccines for Human Papillomavirus (HPV) infection caused cancer such as Gardasil®, Gardasil® and Cervarix®; vaccines that prevent hepatitis B virus caused liver cancer such as Engerix-B® and Recombivax HB®; oncolytic virus therapy that triggers immune response such as Imlygic®; DNA vaccines such as Synchotrope MA2M plasmid DNA vaccine and ZYC101; mammaglobin-a DNA vaccine (see Clinical Cancer Res. 2014 20(23):5964-75); vector based vaccines such as PSA-TRICOM (prostvac), PANVAC-VF, Listeria monocytogenes-based PSA vaccine (see Therapeutic Advances in Vaccines, 2014, 2(5) 137-148), Listeria-mesothelin Adeno-CEA; allogeneic vaccines such as GVAX, BLP-25 (anti-Ankara-mucin 1), Belagenpumatucel-L, TG4010, CIMAvax epidermal growth factor vaccine, NY-ESO, GM.CD40L-CCL21; autologous vaccines such as:Adeno-CD40L, BCG, INGN-225, Dendritic cell vaccines such as Provenge® (Sipuleucel-T), rF-CEA-MUC1-TRICOM (panvac-DC); antigen vaccines such as MUC-1 (stimuvax), NY-ESO-1, GP-100, MAGE-A3 (melanoma antigen encoding gene A3), INGN-225 (see Pharmacology & Therapeutics 153 (2015) 1-9).

In particular embodiments, the antibodies or antigen-binding fragments thereof disclosed herein (e.g., antibody 131A and humanized versions thereof) may be used alone, or in association with chemotherapeutic agents.

In particular embodiments, the antibodies or antigen-binding fragments thereof disclosed herein (e.g., antibody 131A and humanized versions thereof) may be used alone, or in association with radiation therapy.

In particular embodiments, the antibodies or antigen-binding fragments thereof disclosed herein (e.g., antibody 131A and humanized versions thereof) may be used alone, or in association with targeted therapies. Examples of targeted therapies include: hormone therapies, signal transduction inhibitors (e.g., EGFR inhibitors, such as cetuximab (Erbitux) and erlotinib (Tarceva)); HER2 inhibitors (e.g., trastuzumab (Herceptin) and pertuzumab (Perjeta)); BCR-ABL inhibitors (such as imatinib (Gleevec) and dasatinib (Sprycel)); ALK inhibitors (such as crizotinib (Xalkori) and ceritinib (Zykadia)); BRAF inhibitors (such as vemurafenib (Zelboraf) and dabrafenib (Tafinlar)), gene expression modulators, apoptosis inducers (e.g., bortezomib (Velcade) and carfilzomib (Kyprolis)), angiogenesis inhibitors (e.g., bevacizumab (Avastin) and ramucirumab (Cyramza), monoclonal antibodies attached to toxins (e.g., brentuximab vedotin (Adcetris) and ado-trastuzumab emtansine (Kadcyla)).

In particular embodiments, the anti-CD27 antibodies or antigen-binding fragments thereof of the invention (e.g., antibody 131A and humanized versions thereof) may be used in combination with an anti-cancer therapeutic agent or immunomodulatory drug such as an immunomodulatory receptor inhibitor, e.g., an antibody or antigen-binding fragment thereof that specifically binds to the receptor.

In an embodiment of the invention, an anti-CD27 antibody or antigen-binding fragment thereof of the invention (e.g., antibody 131A and humanized versions thereof) is used in association with one or more of: anti-PD1 antibody, anti-PDL1 antibody, anti-TIGIT antibody, anti-CTLA4 antibody, anti-CS1 antibody (e.g., elotuzumab), anti-KIR2DL1/2/3 antibody (e.g., lirilumab), anti-CD137 antibody (e.g., urelumab), anti-GITR antibody (e.g., TRX518), anti-PD1 antibody (e.g., pembrolizumab, nivolumab, pidilizumab (CT-011)), anti-PD-L1 antibody (e.g., BMS-936559, Durvalumab, MSB0010718C or MPDL3280A), anti-PD-L2 antibody, anti-ILT1 antibody, anti-ILT2 antibody, anti-ILT3 antibody, anti-ILT4 antibody, anti-ILT5 antibody, anti-ILT6 antibody, anti-ILT7 antibody, anti-ILT8 antibody, anti-CD40 antibody, anti-OX40 antibody, anti-ICOS, anti-SIRPα, anti-KIR2DL1 antibody, anti-KIR2DL2/3 antibody, anti-KIR2DL4 antibody, anti-KIR2DL5A antibody, anti-KIR2DL5B antibody, anti-KIR3DL1 antibody, anti-KIR3DL2 antibody, anti-KIR3DL3 antibody, anti-NKG2A antibody, anti-NKG2C antibody, anti-NKG2E antibody, anti-4-1BB antibody (e.g., PF-05082566), anti-TSLP antibody, anti-IL-10 antibody, IL-10 or PEGylated IL-10, or any small organic molecule inhibitor of such targets.

In an embodiment of the invention, an anti-CD27 antibody or antigen-binding fragment thereof of the invention (e.g., antibody 131A or a humanized version thereof) is used in association with an anti-PD1 antibody (e.g., pembrolizumab, nivolumab, pidilizumab (CT-011)).

In an embodiment of the invention, an anti-CD27 antibody or antigen-binding fragment thereof of the invention (e.g., antibody 131A or a humanized version thereof) is used in association with an anti-PDL1 antibody (e.g., BMS-936559, Durvalumab, MSB0010718C or MPDL3280A).

In an embodiment of the invention, an anti-CD27 antibody or antigen-binding fragment thereof of the invention (e.g., antibody 131A or a humanized version thereof) is used in association with an anti-CTLA4 antibody.

In an embodiment of the invention, an anti-CD27 antibody or antigen-binding fragment thereof of the invention (e.g., antibody 131A or a humanized version thereof) is used in association with an anti-CS1 antibody.

In an embodiment of the invention, an anti-CD27 antibody or antigen-binding fragment thereof of the invention (e.g., antibody 131A or a humanized version thereof) is used in association with an anti-KIR2DL1/2/3 antibody.

In an embodiment of the invention, an anti-CD27 antibody or antigen-binding fragment thereof of the invention (e.g., antibody 131A or a humanized version thereof) is used in association with an anti-CD137 (e.g., urelumab) antibody.

In an embodiment of the invention, an anti-CD27 antibody or antigen-binding fragment thereof of the invention (e.g., antibody 131A or a humanized version thereof) is used in association with an anti-GITR (e.g., TRX518) antibody.

In an embodiment of the invention, an anti-CD27 antibody or antigen-binding fragment thereof of the invention (e.g., antibody 131A or a humanized version thereof) is used in association with an anti-PD-L2 antibody.

In an embodiment of the invention, an anti-CD27 antibody or antigen-binding fragment thereof of the invention (e.g., antibody 131A or a humanized version thereof) is used in association with an anti-ITL1 antibody.

In an embodiment of the invention, an anti-CD27 antibody or antigen-binding fragment thereof of the invention (e.g., antibody 131A or a humanized version thereof) is used in association with an anti-ITL2 antibody.

In an embodiment of the invention, an anti-CD27 antibody or antigen-binding fragment thereof of the invention (e.g., antibody 131A or a humanized version thereof) is used in association with an anti-ITL3 antibody.

In an embodiment of the invention, an anti-CD27 antibody or antigen-binding fragment thereof of the invention (e.g., antibody 131A or a humanized version thereof) is used in association with an anti-ITL4 antibody.

In an embodiment of the invention, an anti-CD27 antibody or antigen-binding fragment thereof of the invention (e.g., antibody 131A or a humanized version thereof) is used in association with an anti-ITL5 antibody.

In an embodiment of the invention, an anti-CD27 antibody or antigen-binding fragment thereof of the invention (e.g., antibody 131A or a humanized version thereof) is used in association with an anti-ITL6 antibody.

In an embodiment of the invention, an anti-CD27 antibody or antigen-binding fragment thereof of the invention (e.g., antibody 131A or a humanized version thereof) is used in association with an anti-ITL7 antibody.

In an embodiment of the invention, an anti-CD27 antibody or antigen-binding fragment thereof of the invention (e.g., antibody 131A or a humanized version thereof) is used in association with an anti-ITL8 antibody.

In an embodiment of the invention, an anti-CD27 antibody or antigen-binding fragment thereof of the invention (e.g., antibody 131A or a humanized version thereof) is used in association with an anti-CD40 antibody.

In an embodiment of the invention, an anti-CD27 antibody or antigen-binding fragment thereof of the invention (e.g., antibody 131A or a humanized version thereof) is used in association with an anti-OX40 antibody.

In an embodiment of the invention, an anti-CD27 antibody or antigen-binding fragment thereof of the invention (e.g., antibody 131A or a humanized version thereof) is used in association with an anti-KIR2DL1 antibody.

In an embodiment of the invention, an anti-CD27 antibody or antigen-binding fragment thereof of the invention (e.g., antibody 131A or a humanized version thereof) is used in association with an anti-KIR2DL2/3 antibody.

In an embodiment of the invention, an anti-CD27 antibody or antigen-binding fragment thereof of the invention (e.g., antibody 131A or a humanized version thereof) is used in association with an anti-KIR2DL4 antibody.

In an embodiment of the invention, an anti-CD27 antibody or antigen-binding fragment thereof of the invention (e.g., antibody 131A or a humanized version thereof) is used in association with an anti-KIR2DL5A antibody.

In an embodiment of the invention, an anti-CD27 antibody or antigen-binding fragment thereof of the invention (e.g., antibody 131A or a humanized version thereof) is used in association with an anti-KIR2DL5B antibody.

In an embodiment of the invention, an anti-CD27 antibody or antigen-binding fragment thereof of the invention (e.g., antibody 131A or a humanized version thereof) is used in association with an anti-KIR3DL1 antibody.

In an embodiment of the invention, an anti-CD27 antibody or antigen-binding fragment thereof of the invention (e.g., antibody 131A or a humanized version thereof) is used in association with an anti-KIR3DL2 antibody.

In an embodiment of the invention, an anti-CD27 antibody or antigen-binding fragment thereof of the invention (e.g., antibody 131A or a humanized version thereof) is used in association with an anti-KIR3DL3 antibody.

In an embodiment of the invention, an anti-CD27 antibody or antigen-binding fragment thereof of the invention (e.g., antibody 131A or a humanized version thereof) is used in association with an anti-NKG2A antibody.

In an embodiment of the invention, an anti-CD27 antibody or antigen-binding fragment thereof of the invention (e.g., antibody 131A or a humanized version thereof) is used in association with an anti-NKG2C antibody.

In an embodiment of the invention, an anti-CD27 antibody or antigen-binding fragment thereof of the invention (e.g., antibody 131A or a humanized version thereof) is used in association with an anti-ICOS antibody.

In an embodiment of the invention, an anti-CD27 antibody or antigen-binding fragment thereof of the invention (e.g., antibody 131A or a humanized version thereof) is used in association with an anti-SIRPα antibody.

In an embodiment of the invention, an anti-CD27 antibody or antigen-binding fragment thereof of the invention (e.g., antibody 131A or a humanized version thereof) is used in association with an anti-4-1BB antibody.

In an embodiment of the invention, an anti-CD27 antibody or antigen-binding fragment thereof of the invention (e.g., antibody 131A or a humanized version thereof) is used in association with an anti-IL-10 antibody.

In an embodiment of the invention, an anti-CD27 antibody or antigen-binding fragment thereof of the invention (e.g., antibody 131A or a humanized version thereof) is used in association with an anti-TSLP antibody.

In an embodiment of the invention, an anti-CD27 antibody or antigen-binding fragment thereof of the invention (e.g., antibody 131A or a humanized version thereof) is used in association with IL-10 or PEGylated IL-10.

In an embodiment of the invention, an anti-CD27 antibody or antigen-binding fragment thereof of the invention (e.g., antibody 131A or a humanized version thereof) is used in association with one or more of an inhibitor (e.g., a small organic molecule or an antibody or antigen-binding fragment thereof) such as: an MTOR (mammalian target of rapamycin) inhibitor, a cytotoxic agent, a platinum agent, an EGFR inhibitor, a VEGF inhibitor, a microtubule stabilizer, a taxane, a CD20 inhibitor, a CD52 inhibitor, a CD30 inhibitor, a RANK (Receptor activator of nuclear factor kappa-B) inhibitor, a STING agonist, a CXCR2 antagonist, a RANKL (Receptor activator of nuclear factor kappa-B ligand) inhibitor, an ERK inhibitor, a MAP Kinase inhibitor, an AKT inhibitor, a MEK inhibitor, a PARP inhibitor, a PI3K inhibitor, a HER1 inhibitor, a HER2 inhibitor, a HER3 inhibitor, a HER4 inhibitor, a Bcl2 inhibitor, a CD22 inhibitor, a CD79b inhibitor, an ErbB2 inhibitor, or a farnesyl protein transferase inhibitor.

In an embodiment of the invention, an anti-CD27 antibody or antigen-binding fragment thereof of the invention (e.g., antibody 131A or a humanized version thereof) is used in association with any one or more of: 13-cis-retinoic acid, 3-[5-(methylsulfonylpiperadinemethyl)-indolyl]-quinolone, 4-hydroxytamoxifen, 5-deooxyuridine, 5′-deoxy-5-fluorouridine, 5-fluorouracil, 6-mecaptopurine, 7-hydroxystaurosporine, A-443654, abirateroneacetate, abraxane, ABT-578, acolbifene, ADS-100380, ALT-110, altretamine, amifostine, aminoglutethimide, amrubicin, Amsacrine, anagrelide, anastrozole, angiostatin, AP-23573, ARQ-197, arzoxifene, AS-252424, AS-605240, asparaginase, AT-9263, atrasentan, axitinib, AZD 1152, Bacillus Calmette-Guerin (BCG) vaccine, batabulin, BC-210, besodutox, bevacizumab, bicalutamide, Biol11, B10140, bleomycin, BMS-214662, BMS-247550, BMS-275291, BMS-310705, bortezimib, buserelin, busulfan, calcitriol, camptothecin, canertinib, capecitabine, carboplatin, carmustine, CC8490, Cediranib, CG-1521, CG-781, chlamydocin, chlorambucil, chlorotoxin, cilengitide, cimitidine, cisplatin, cladribine, clodronate, COL-3, CP-724714, cyclophosphamide, cyproterone, cyproteroneacetate, cytarabine, cytosinearabinoside, dacarbazine, dacinostat, dactinomycin, dalotuzumab, danusertib, dasatanib, daunorubicin, decatanib, deguelin, denileukin, deoxycoformycin, depsipeptide, diarylpropionitrile, diethylstilbestrol, diftitox, docetaxel, dovitinib, doxorubicin, droloxifene, edotecarin, yttrium-90 labeled-edotreotide, edotreotide, EKB-569, EMD121974, endostatin, enzalutamide, enzastaurin, epirubicin, epithilone B, ERA-923, Erbitux, erlotinib, estradiol, estramustine, etoposide, everolimus, exemestane, ficlatuzumab, finasteride, flavopiridol, floxuridine, fludarabine, fludrocortisone, fluoxymesterone, flutamide, FOLFOX regimen, Fulvestrant, galeterone, gefitinib, gemcitabine, gimatecan, goserelin, goserelin acetate, gossypol, GSK461364, GSK690693, HMR-3339, hydroxyprogesteronecaproate, hydroxyurea, IC87114, idarubicin, idoxyfene, ifosfamide, IM862, imatinib, IMC-1C11, INCB24360, INO1001, interferon, interleukin-12, ipilimumab, irinotecan, JNJ-16241199, ketoconazole, KRX-0402, lapatinib, lasofoxifene, letrozole, leucovorin, leuprolide, leuprolide acetate, levamisole, liposome entrapped paclitaxel, lomustine, lonafamib, lucanthone, LY292223, LY292696, LY293646, LY293684, LY294002, LY317615, marimastat, mechlorethamine, medroxyprogesteroneacetate, megestrolacetate, melphalan, mercaptopurine, mesna, methotrexate, mithramycin, mitomycin, mitotane, mitoxantrone, tozasertib, MLN8054, neovastat, Neratinib, neuradiab, nilotinib, nilutimide, nolatrexed, NVP-BEZ235, oblimersen, octreotide, ofatumumab, olaparib, oregovomab, orteronel, oxaliplatin, paclitaxel, palbociclib, pamidronate, panitumumab, pazopanib, PD0325901, PD184352, PEG-interferon, pemetrexed, pentostatin, perifosine, phenylalaninemustard, PI-103, pictilisib, PIK-75, pipendoxifene, PKI-166, plicamycin, porfimer, prednisone, procarbazine, progestins, PX-866, R-763, raloxifene, raltitrexed, razoxin, ridaforolimus, rituximab, romidepsin, RTA744, rubitecan, scriptaid, Sdx102, seliciclib, selumetinib, semaxanib, SF1126, sirolimus, SN36093, sorafenib, spironolactone, squalamine, SR13668, streptozocin, SU6668, suberoylanalide hydroxamic acid, sunitinib, synthetic estrogen, talampanel, talimogene laherparepvec, tamoxifen, temozolomide, temsirolimus, teniposide, tesmilifene, testosterone, tetrandrine, TGX-221, thalidomide, thioguanine, thiotepa, ticilimumab, tipifamib, tivozanib, TKI-258, TLK286, topotecan, toremifene citrate, trabectedin, trastuzumab, tretinoin, trichostatin A, triciribinephosphate monohydrate, triptorelin pamoate, TSE-424, uracil mustard, valproic acid, valrubicin, vandetanib, vatalanib, VEGF trap, vinblastine, vincristine, vindesine, vinorelbine, vitaxin, vitespan, vorinostat, VX-745, wortmannin, Xr311, zanolimumab, ZK186619, ZK-304709, ZM336372, ZSTK474.

Non-limiting examples of suitable anti-cancer agents to be used in combination with an anti-CD27 antibody or antigen-binding fragment thereof of the invention include cytostatic agents, cytotoxic agents, targeted therapeutic agents (small molecules, biologics, siRNA and microRNA) against cancer and neoplastic diseases,

-   -   1) anti-metabolites (such as methoxtrexate, 5-fluorouracil,         gemcitabine, fludarabine, capecitabine);     -   2) alkylating agents, such as temozolomide, cyclophosphamide,     -   3) DNA interactive and DNA damaging agents, such as cisplatin,         oxaliplatin, doxorubicin,     -   4) Ionizing irradiation, such as radiation therapy,     -   5) topoisomerase II inhibitors, such as etoposide, doxorubicin,     -   6) topoisomerase I inhibitors, such as irinotecan, topotecan,     -   7) tubulin interacting agents, such as paclitaxel, docetaxel,         Abraxane, epothilones,     -   8) kinesin spindle protein inhibitors,     -   9) spindle checkpoint inhibitors,     -   10) Poly(ADP-ribose) polymerase (PARP) inhibitors, such as         olaparib, niraparib and veliparib     -   11) Matrix metalloprotease (MMP) inhibitors     -   12) Protease inhibitors, such as cathepsin D and cathepsin K         inhibitors     -   13) Proteosome or ubiquitination inhibitors, such as bortezomib,     -   14) Activator of mutant p53 to restore its wild-type p53         activity     -   15) Adenoviral-p53     -   16) Bcl-2 inhibitors, such as ABT-263     -   17) Heat shock protein (HSP) modulators, such as geldanamycin         and 17-AAG     -   18) Histone deacetylase (HDAC) inhibitors, such as vorinostat         (SAHA),     -   19) sex hormone modulating agents,         -   a. anti-estrogens, such as tamoxifen, fulvestrant,         -   b. selective estrogen receptor modulators (SERM), such as             raloxifene,         -   c. anti-androgens, such as bicalutamide, flutamide         -   d. LHRH agonists, such as leuprolide,         -   e. 5c-reductase inhibitors, such as finasteride,         -   f. Cytochrome P450 C17 lyase (CYP450c17, also called 17aC);         -   g. aromatase inhibitors, such as letrozole, anastrozole,             exemestane,     -   20) EGFR kinase inhibitors, such as geftinib, erlotinib,         laptinib     -   21) dual erbB1 and erbB2 inhibitors, such as Lapatinib     -   22) multi-targeted kinases (serine/threonine and/or tyrosine         kinase) inhibitors,         -   a. ABL kinase inhibitors, imatinib and nilotinib, dasatinib         -   b. VEGFR-1, VEGFR-2, PDGFR, KDR, FLT, c-Kit, Tie2, Raf, MEK             and ERK inhibitors, such as sunitinib, sorafenib,             Vandetanib, pazopanib, PLX-4032, Axitinib, PTK787,             GSK-1120212         -   c. Polo-like kinase inhibitors         -   d. Aurora kinase inhibitors         -   e. JAK inhibitor         -   f. c-MET kinase inhibitors         -   g. PI3K and mTOR inhibitors, such as GDC-0941, BEZ-235,             BKM-120 and AZD-8055         -   h. Rapamycin and its analogs, such as Temsirolimus,             everolimus, and deforolimus         -   i. STING (Stimulator of Interferon Genes) agonist         -   j. CXCR (CXC Chemokine Receptor) inhibitor, CXCR2 antagonist     -   23) and other anti-cancer (also know as anti-neoplastic) agents         include but are not limited to ara-C, adriamycin, cytoxan,         Carboplatin, Uracil mustard, Clormethine, Ifosfsmide, Melphalan,         Chlorambucil, Pipobroman, Triethylenemelamine,         Triethylenethiophosphoramine, Busulfan, Carmustine, Lomustine,         Streptozocin, Dacarbazine, Floxuridine, Cytarabine,         6-Mercaptopurine, 6-Thioguanine, Fludarabine phosphate,         Pentostatine, Vinblastine, Vincristine, Vindesine, Vinorelbine,         Navelbine, Bleomycin, Dactinomycin, Daunorubicin, Doxorubicin,         Epirubicin, teniposide, cytarabine, pemetrexed, Idarubicin,         Mithramycin, Deoxycoformycin, Mitomycin-C, L-Asparaginase,         Teniposide, Ethinylestradiol, Diethylstilbestrol, Testosterone,         Prednisone, Fluoxymesterone, Dromostanolone propionate,         Testolactone, Megestrolacetate, Methylprednisolone,         Methyltestosterone, Prednisolone, Triamcinolone,         Chlorotrianisene, Hydroxyprogesterone, Aminoglutethimide,         Estramustine, Flutamide Medroxyprogesteroneacetate, Toremifene,         goserelin, Carboplatin, Hydroxyurea, Amsacrine, Procarbazine,         Mitotane, Mitoxantrone, Levamisole, Drolloxafine,         Hexamethylmelamine, Bexxar, Zevalin, Trisenox, Profimer,         Thiotepa, Altretamine, Doxil, Ontak, Depocyt, Aranesp, Neupogen,         Neulasta, Kepivance.     -   24) Farnesyl protein transferase inhibitors, such as, SARASAR™         (4-[2-[4-[(11R)-3,10-dibromo-8-chloro-6,         11-dihydro-5H-benzo[5,6]cyclohepta[1,2-b]pyridin-11-yl-]-1-piperidinyl]-2-oxoethyl]-piperidinecarboxamide,         tipifamib     -   25) interferons, such as Intron A, Peg-Intron,     -   26) anti-erbB1 antibodies, such as cetuximab, panitumumab,     -   27) anti-erbB2 antibodies, such as trastuzumab,     -   28) anti-CD52 antibodies, such as Alemtuzumab,     -   29) anti-CD20 antibodies, such as Rituximab     -   30) anti-CD33 antibodies, such as Gemtuzumab ozogamicin     -   31) anti-VEGF antibodies, such as Avastin,     -   32) TRIAL ligands, such as Lexatumumab, mapatumumab, and AMG-655     -   33) anti-CTLA-4 antibodies, such as ipilimumab     -   34) antibodies against CTA1, CEA, CD5, CD19, CD22, CD30, CD44,         CD44V6, CD55, CD56, EpCAM, FAP, MHCII, HGF, IL-6, MUC1, PSMA,         TAL6, TAG-72, TRAILR, VEGFR, IGF-2, FGF,     -   35) anti-IGF-1R antibodies, such as dalotuzumab (MK-0646) and         robatumumab (SCH 717454).

“Estrogen receptor modulators” refers to compounds that interfere with or inhibit the binding of estrogen to the receptor, regardless of mechanism. Examples of estrogen receptor modulators include, but are not limited to, tamoxifen, raloxifene, idoxifene, LY353381, LY117081, toremifene, fulvestrant, 4-[7-(2,2-dimethyl-1-oxopropoxy-4-methyl-2-[4-[2-(1-piperidinyl)ethoxy]phenyl]-2H-1-benzopyran-3-yl]-phenyl-2,2-dimethylpropanoate, 4,4′-dihydroxybenzophenone-2,4-dinitrophenyl-hydrazone, and SH646.

“Androgen receptor modulators” refers to compounds which interfere or inhibit the binding of androgens to the receptor, regardless of mechanism. Examples of androgen receptor modulators include finasteride and other 5a-reductase inhibitors, nilutamide, flutamide, bicalutamide, liarozole, and abiraterone acetate.

“Retinoid receptor modulators” refers to compounds which interfere or inhibit the binding of retinoids to the receptor, regardless of mechanism. Examples of such retinoid receptor modulators include bexarotene, tretinoin, 13-cis-retinoic acid, 9-cis-retinoic acid, a-difluoromethylomithine, ILX23-7553, trans-N-(4′-hydroxyphenyl) retinamide, and N-4-carboxyphenyl retinamide.

“Cytotoxic/cytostatic agents” refer to compounds which cause cell death or inhibit cell proliferation primarily by interfering directly with the cell's functioning or inhibit or interfere with cell myosis, including alkylating agents, tumor necrosis factors, intercalators, hypoxia activatable compounds, microtubule inhibitors/microtubule-stabilizing agents, inhibitors of mitotic kinesins, histone deacetylase inhibitors, inhibitors of kinases involved in mitotic progression, inhibitors of kinases involved in growth factor and cytokine signal transduction pathways, antimetabolites, biological response modifiers, hormonal/anti-hormonal therapeutic agents, haematopoietic growth factors, monoclonal antibody targeted therapeutic agents, topoisomerase inhibitors, proteosome inhibitors, ubiquitin ligase inhibitors, and aurora kinase inhibitors.

Examples of cytotoxic/cytostatic agents include, but are not limited to, platinum coordinator compounds, sertenef, cachectin, ifosfamide, tasonermin, lonidamine, carboplatin, altretamine, prednimustine, dibromodulcitol, ranimustine, fotemustine, nedaplatin, oxaliplatin, temozolomide, heptaplatin, estramustine, improsulfan tosilate, trofosfamide, nimustine, dibrospidium chloride, pumitepa, lobaplatin, satraplatin, profiromycin, cisplatin, irofulven, dexifosfamide, cis-aminedichloro(2-methyl-pyridine)platinum, benzylguanine, glufosfamide, GPX100, (trans, trans, trans)-bis-mu-(hexane-1,6-diamine)-mu-[diamine-platinum(II)]bis[diamine(chloro)platinum (II)]tetrachloride, diarizidinylspermine, arsenic trioxide, 1-(11-dodecylamino-10-hydroxyundecyl)-3,7-dimethylxanthine, zorubicin, idarubicin, daunorubicin, bisantrene, mitoxantrone, pirarubicin, pinafide, valrubicin, amrubicin, antineoplaston, 3′-deamino-3′-morpholino-13-deoxo-10-hydroxycarminomycin, annamycin, galarubicin, elinafide, MEN10755, 4-demethoxy-3-deamino-3-aziridinyl-4-methylsulphonyl-daunorubicin (see WO 00/50032).

An example of a hypoxia activatable compound is tirapazamine.

Examples of proteosome inhibitors include but are not limited to lactacystin and MLN-341 (Velcade).

Examples of microtubule inhibitors/microtubule-stabilising agents include taxanes in general. Specific compounds include paclitaxel (Taxol®), vindesine sulfate, 3′,4′-didehydro-4′-deoxy-8′-norvincaleukoblastine, docetaxol (Taxotere®), rhizoxin, dolastatin, mivobulin isethionate, auristatin, cemadotin, RPR109881, BMS 184476, vinflunine, cryptophycin, 2,3,4,5,6-pentafluoro-N-(3-fluoro-4-methoxyphenyl) benzene sulfonamide, anhydrovinblastine, N,N-dimethyl-L-valyl-L-valyl-N-methyl-L-valyl-L-prolyl-L-proline-t-butylamide, (SEQ ID NO: 68), TDX258, the epothilones (see for example U.S. Pat. Nos. 6,284,781 and 6,288,237) and BMS 188797.

Some examples of topoisomerase inhibitors are topotecan, hycaptamine, irinotecan, rubitecan, 6-ethoxypropionyl-3′,4′-O-exo-benzylidene-chartreusin, 9-methoxy-N,N-dimethyl-5-nitropyrazolo[3,4,5-kl]acridine-2-(6H) propanamine, 1-amino-9-ethyl-5-fluoro-2,3-dihydro-9-hydroxy-4-methyl-1H, 12H-benzo[de]pyrano[3′,4′:b,7]-indolizino[1,2b]quinoline-10,13(9H,15H)dione, lurtotecan, 7-[2-(N-isopropylamino)ethyl]-(20S)camptothecin, BNP1350, BNPI1100, BN80915, BN80942, etoposide phosphate, teniposide, sobuzoxane, 2′-dimethylamino-2′-deoxy-etoposide, GL331, N-[2-(dimethylamino)ethyl]-9-hydroxy-5,6-dimethyl-6H-pyrido[4,3-b]carbazole-1-carboxamide, asulacrine, (5a, 5aB, 8aa,9b)-9-[2-[N-[2-(dimethylamino)ethyl]-N-methylamino]ethyl]-5-[4-hydroxy-3,5-dimethoxyphenyl]-5,5a,6,8,8a,9-hexohydrofuro(3′,4′: 6,7)naphtho(2,3-d)-1,3-dioxol-6-one, 2,3-(methylenedioxy)-5-methyl-7-hydroxy-8-methoxybenzo[c]-phenanthridinium, 6,9-bis[(2-aminoethyl)amino]benzo[g]isoguinoline-5,10-dione, 5-(3-aminopropylamino)-7,10-dihydroxy-2-(2-hydroxyethylaminomethyl)-6H-pyrazolo[4,5,1-de]acridin-6-one, N-[1-[2(diethylamino)ethylamino]-7-methoxy-9-oxo-9H-thioxanthen-4-ylmethyl]formamide, N-(2-(dimethylamino)ethyl)acridine-4-carboxamide, 6-[[2-(dimethylamino)ethyl]amino]-3-hydroxy-7H-indeno[2,1-c] quinolin-7-one, and dimesna.

Examples of inhibitors of mitotic kinesins, and in particular the human mitotic kinesin KSP, are described in Publications WO03/039460, WO03/050064, WO03/050122, WO03/049527, WO03/049679, WO03/049678, WO04/039774, WO03/079973, WO03/099211, WO03/105855, WO03/106417, WO04/037171, WO04/058148, WO004/058700, WO04/126699, WO05/018638, WO005/019206, WO005/019205, WO05/018547, WO05/017190, US2005/0176776. In an embodiment inhibitors of mitotic kinesins include, but are not limited to inhibitors of KSP, inhibitors of MKLP1, inhibitors of CENP-E, inhibitors of MCAK and inhibitors of Rab6-KIFL.

Examples of “histone deacetylase inhibitors” include, but are not limited to, SAHA, TSA, oxamflatin, PXD101, MG98 and scriptaid. Further reference to other histone deacetylase inhibitors may be found in the following manuscript; Miller, T. A. et al. J. Med. Chem. 46(24):5097-5116 (2003).

“Inhibitors of kinases involved in mitotic progression” include, but are not limited to, inhibitors of aurora kinase, inhibitors of Polo-like kinases (PLK; in particular inhibitors of PLK-1), inhibitors of bub-1 and inhibitors of bub-R1. An example of an “aurora kinase inhibitor” is VX-680.

“Antiproliferative agents” includes antisense RNA and DNA oligonucleotides such as G3139, ODN698, RVASKRAS, GEM231, and INX3001, and antimetabolites such as enocitabine, carmofur, tegafur, pentostatin, doxifluridine, trimetrexate, fludarabine, capecitabine, galocitabine, cytarabine ocfosfate, fosteabine sodium hydrate, raltitrexed, paltitrexid, emitefur, tiazofurin, decitabine, nolatrexed, pemetrexed, nelzarabine, 2′-deoxy-2′-methylidenecytidine, 2′-fluoromethylene-2′-deoxycytidine, N-[5-(2,3-dihydro-benzofuryl)sulfonyl]-N′-(3,4-dichlorophenyl)urea, N6-[4-deoxy-4-[N2-[2(E),4(E)-tetradecadienoyl]glycylamino]-L-glycero-B-L-manno-heptopyranosyl]adenine, aplidine, ecteinascidin, troxacitabine, 4-[2-amino-4-oxo-4,6,7,8-tetrahydro-3H-pyrimidino[5,4-b][1,4]thiazin-6-yl-(S)-ethyl]-2,5-thienoyl-L-glutamic acid, aminopterin, 5-flurouracil, alanosine, 11-acetyl-8-(carbamoyloxymethyl)-4-formyl-6-methoxy-14-oxa-1,11-diazatetracyclo(7.4.1.0.0)-tetradeca-2,4,6-trien-9-yl acetic acid ester, swainsonine, lometrexol, dexrazoxane, methioninase, 2′-cyano-2′-deoxy-N4-palmitoyl-1-B-D-arabino furanosyl cytosine, 3-aminopyridine-2-carboxaldehyde thiosemicarbazone and trastuzumab.

Examples of monoclonal antibody targeted therapeutic agents include those therapeutic agents which have cytotoxic agents or radioisotopes attached to a cancer cell specific or target cell specific monoclonal antibody. Examples include Bexxar.

“Prenyl-protein transferase inhibitor” refers to a compound which inhibits any one or any combination of the prenyl-protein transferase enzymes, including famesyl-protein transferase (FPTase), geranylgeranyl-protein transferase type I (GGPTase-I), and geranylgeranyl-protein transferase type-II (GGPTase-II, also called Rab GGPTase).

Examples of prenyl-protein transferase inhibitors can be found in the following publications and patents: WO 96/30343, WO 97/18813, WO 97/21701, WO 97/23478, WO 97/38665, WO 98/28980, WO 98/29119, WO 95/32987, U.S. Pat. Nos. 5,420,245, 5,523,430, 5,532,359, 5,510,510, 5,589,485, 5,602,098, European Patent Publ. 0 618 221, European Patent Publ. 0 675 112, European Patent Publ. 0 604 181, European Patent Publ. 0 696 593, WO 94/19357, WO 95/08542, WO 95/11917, WO 95/12612, WO 95/12572, WO 95/10514, U.S. Pat. No. 5,661,152, WO 95/10515, WO 95/10516, WO 95/24612, WO 95/34535, WO 95/25086, WO 96/05529, WO 96/06138, WO 96/06193, WO 96/16443, WO 96/21701, WO 96/21456, WO 96/22278, WO 96/24611, WO 96/24612, WO 96/05168, WO 96/05169, WO 96/00736, U.S. Pat. No. 5,571,792, WO 96/17861, WO 96/33159, WO 96/34850, WO 96/34851, WO 96/30017, WO 96/30018, WO 96/30362, WO 96/30363, WO 96/31111, WO 96/31477, WO 96/31478, WO 96/31501, WO 97/00252, WO 97/03047, WO 97/03050, WO 97/04785, WO 97/02920, WO 97/17070, WO 97/23478, WO 97/26246, WO 97/30053, WO 97/44350, WO 98/02436, and U.S. Pat. No. 5,532,359. For an example of the role of a prenyl-protein transferase inhibitor on angiogenesis see European J. of Cancer, Vol. 35, No. 9, pp. 1394-1401 (1999).

“Angiogenesis inhibitors” refers to compounds that inhibit the formation of new blood vessels, regardless of mechanism. Examples of angiogenesis inhibitors include, but are not limited to, tyrosine kinase inhibitors, such as inhibitors of the tyrosine kinase receptors Flt-1 (VEGFR1) and Flk-1/KDR (VEGFR2), inhibitors of epidermal-derived, fibroblast-derived, or platelet derived growth factors, MMP (matrix metalloprotease) inhibitors, integrin blockers, interferon-a, interleukin-12, pentosan polysulfate, cyclooxygenase inhibitors, including nonsteroidal anti-inflammatories (NSAIDs) like aspirin and ibuprofen as well as selective cyclooxy-genase-2 inhibitors like celecoxib and rofecoxib (PNAS, Vol. 89, p. 7384 (1992); JNCI, Vol. 69, p. 475 (1982); Arch. Opthalmol., Vol. 108, p. 573 (1990); Anat. Rec., Vol. 238, p. 68 (1994); FEBS Letters, Vol. 372, p. 83 (1995); Clin, Orthop. Vol. 313, p. 76 (1995); J. Mol. Endocrinol., Vol. 16, p. 107 (1996); Jpn. J. Pharmacol., Vol. 75, p. 105 (1997); Cancer Res., Vol. 57, p. 1625 (1997); Cell, Vol. 93, p. 705 (1998); Intl. J. Mol. Med., Vol. 2, p. 715 (1998); J. Biol. Chem., Vol. 274, p. 9116 (1999)), steroidal anti-inflammatories (such as corticosteroids, mineralocorticoids, dexamethasone, prednisone, prednisolone, methylpred, betamethasone), carboxyamidotriazole, combretastatin A-4, squalamine, 6-O-chloroacetyl-carbonyl)-fumagillol, thalidomide, angiostatin, troponin-1, angiotensin II antagonists (see Fernandez et al., J. Lab. Clin. Med. 105:141-145 (1985)), and antibodies to VEGF (see, Nature Biotechnology, Vol. 17, pp. 963-968 (October 1999); Kim et al., Nature, 362, 841-844 (1993); WO 00/44777; and WO 00/61186).

Other examples of angiogenesis inhibitors include, but are not limited to, endostatin, ukrain, ranpimase, IM862, 5-methoxy-4-[2-methyl-3-(3-methyl-2-butenyl)oxiranyl]-1-oxaspiro[2,5]oct-6-yl (chloroacetyl)carbamate, acetyldinanaline, 5-amino-1-[[3,5-dichloro-4-(4-chlorobenzoyl)phenyl]methyl]-1H-1,2,3-triazole-4-carboxamide, CM101, squalamine, combretastatin, RPI4610, NX31838, sulfated mannopentaose phosphate, 7,7-(carbonyl-bis [imino-N-methyl-4,2-pyrrolocarbonylimino[N-methyl-4,2-pyrrole]-carbonylimino]-bis-(1,3-naphthalene disulfonate), and 3-[(2,4-dimethylpyrrol-5-yl)methylene]-2-indolinone (SU5416).

Other therapeutic agents that modulate or inhibit angiogenesis and may also be used in combination with the compounds of the instant invention include agents that modulate or inhibit the coagulation and fibrinolysis systems (see review in Clin. Chem. La. Med. 38:679-692 (2000)). Examples of such agents that modulate or inhibit the coagulation and fibrinolysis pathways include, but are not limited to, heparin (see Thromb. Haemost. 80:10-23 (1998)), low molecular weight heparins and carboxypeptidase U inhibitors (also known as inhibitors of active thrombin activatable fibrinolysis inhibitor [TAFIa]) (see Thrombosis Res. 101:329-354 (2001)). TAFIa inhibitors have been described in U.S. Ser. Nos. 60/310,927 (filed Aug. 8, 2001) and 60/349,925 (filed Jan. 18, 2002).

“Agents that interfere with receptor tyrosine kinases (RTKs)” refer to compounds that inhibit RTKs and therefore mechanisms involved in oncogenesis and tumor progression. Such agents include inhibitors of c-Kit, Eph, PDGF, Flt3 and c-Met. Further agents include inhibitors of RTKs as described by Bume-Jensen and Hunter, Nature, 411:355-365, 2001.

“Inhibitors of cell proliferation and survival signalling pathway” refer to compounds that inhibit signal transduction cascades downstream of cell surface receptors. Such agents include inhibitors of serine/threonine kinases (including but not limited to inhibitors of Akt such as described in WO 02/083064, WO 02/083139, WO 02/083140, US 2004-0116432, WO 02/083138, US 2004-0102360, WO 03/086404, WO 03/086279, WO 03/086394, WO 03/084473, WO 03/086403, WO 2004/041162, WO 2004/096131, WO 2004/096129, WO 2004/096135, WO 2004/096130, WO 2005/100356, WO 2005/100344, US 2005/029941, US 2005/44294, US 2005/43361, 60/734188, 60/652737, 60/670469), inhibitors of Raf kinase (for example PLX-4032), inhibitors of MEK (for example Arry-162, RO-4987655 and GSK-1120212), inhibitors of mTOR (for example AZD-8055, BEZ-235 and everolimus), and inhibitors of PI3K (for example GDC-0941, BKM-120).

As used above, “integrin blockers” refers to compounds which selectively antagonize, inhibit or counteract binding of a physiological ligand to the α_(v)β₃ integrin, to compounds which selectively antagonize, inhibit or counteract binding of a physiological ligand to the α_(v)β5 integrin, to compounds which antagonize, inhibit or counteract binding of a physiological ligand to both the α_(v)β₃ integrin and the α_(v)β₅ integrin, and to compounds which antagonize, inhibit or counteract the activity of the particular integrin(s) expressed on capillary endothelial cells. The term also refers to antagonists of the α_(v)β₆, α_(v)β₂, α₁β₁, α₂β₁, α₅β₁, α₆β₁, and α₆β₄, integrins. The term also refers to antagonists of any combination of α_(v)β₃, α_(v)β₅, α_(v)β₆, α_(v)β₈, α₁β₁, α₂β₁, α₆β₁, α₆β₁ and α₆β₄ integrins.

Some specific examples of tyrosine kinase inhibitors include N-(trifluoromethylphenyl)-5-methylisoxazol-4-carboxamide, 3-[(2,4-dimethylpyrrol-5-yl)methylidenyl)indolin-2-one, 17-(allylamino)-17-demethoxygeldanamycin, 4-(3-chloro-4-fluorophenylamino)-7-methoxy-6-[3-(4-morpholinyl)propoxyl]quinazoline, N-(3-ethynylphenyl)-6,7-bis(2-methoxyethoxy)-4-quinazolinamine, BIBX1382, 2,3,9,10,11,12-hexahydro-10-(hydroxymethyl)-10-hydroxy-9-methyl-9,12-epoxy-1H-diindolo[1,2,3-fg:3′,2′,1′-kl]pyrrolo[3,4-i][1,6]benzodiazocin-1-one, SH268, genistein, STI571, CEP2563, 4-(3-chlorophenylamino)-5,6-dimethyl-7H-pyrrolo[2,3-d]pyrimidinemethane sulfonate, 4-(3-bromo-4-hydroxyphenyl)amino-6,7-dimethoxyquinazoline, 4-(4′-hydroxyphenyl)amino-6,7-dimethoxyquinazoline, SU6668, STI571A, N-4-chlorophenyl-4-(4-pyridylmethyl)-1-phthalazinamine, and EMD121974.

Combinations of the instantly claimed antibodies or antigen binding fragments with PPAR-γ (i.e., PPAR-gamma) agonists and PPAR-δ (i.e., PPAR-delta) agonists may be useful in the treatment of certain malignancies. PPAR-γ and PPAR-δ are the nuclear peroxisome proliferator-activated receptors γ and δ. The expression of PPAR-γ on endothelial cells and its involvement in angiogenesis has been reported in the literature (see J. Cardiovasc. Pharmacol. 1998; 31:909-913; J. Biol. Chem. 1999; 274:9116-9121; Invest. Ophthalmol Vis. Sci. 2000; 41:2309-2317). More recently, PPAR-γ agonists have been shown to inhibit the angiogenic response to VEGF in vitro; both troglitazone and rosiglitazone maleate inhibit the development of retinal neovascularization in mice. (Arch. Ophthamol. 2001; 119:709-717). Examples of PPAR-γ agonists and PPAR-γ/α agonists include, but are not limited to, Lynparza®, Rucaparib®, Talazoparib®, niraparib, Veliparib®, thiazolidinediones (such as DRF2725, CS-011, troglitazone, rosiglitazone, and pioglitazone), fenofibrate, gemfibrozil, clofibrate, GW2570, SB219994, AR-H039242, JTT-501, MCC-555, GW2331, GW409544, NN2344, KRP297, NP0110, DRF4158, NN622, G1262570, PNU182716, DRF552926, 2-[(5,7-dipropyl-3-trifluoromethyl-1,2-benzisoxazol-6-yl)oxy]-2-methylpropionic acid, and 2(R)-7-(3-(2-chloro-4-(4-fluorophenoxy) phenoxy)propoxy)-2-ethylchromane-2-carboxylic acid.

The antibody or antigen binding fragment of the instant invention may also be useful for treating or preventing breast cancer in combination with aromatase inhibitors. Examples of aromatase inhibitors include but are not limited to: anastrozole, letrozole and exemestane.

The antibody or antigen binding fragment of the instant invention may also be useful for treating cancer in combination with the following chemotherapeutic agents: abarelix (Plenaxis Depot®); aldesleukin (Prokine®); Aldesleukin (Proleukin®); Alemtuzumabb (Campath®); alitretinoin (Panretin®); allopurinol (Zyloprim®); altretamine (Hexalen®); amifostine (Ethyol®); anastrozole (Arimidex®); arsenic trioxide (Trisenox®); asparaginase (Elspar®); azacitidine (Vidaza®); bendamustine hydrochloride (Treanda®); bevacuzimab (Avastin®); bexarotene capsules (Targretin®); bexarotene gel (Targretin®); bleomycin (Blenoxane®); bortezomib (Velcade®); brefeldin A; busulfan intravenous (Busulfex®); busulfan oral (Myleran®); calusterone (Methosarb®); capecitabine (Xeloda®); carboplatin (Paraplatin®); carmustine (BCNU®, BiCNU®); carmustine (Gliadel®); carmustine with Polifeprosan 20 Implant (Gliadel Wafer®); celecoxib (Celebrex®); cetuximab (Erbitux®); chlorambucil (Leukeran®); cisplatin (Platinol®); cladribine (Leustatin®, 2-CdA®); clofarabine (Clolar®); cyclophosphamide (Cytoxan®, Neosar®); cyclophosphamide (Cytoxan Injection®); cyclophosphamide (Cytoxan Tablet®); cytarabine (Cytosar-U®); cytarabine liposomal (DepoCyt®); dacarbazine (DTIC-Dome®); dactinomycin, actinomycin D (Cosmegen®); dalteparin sodium injection (Fragmin®); Darbepoetin alfa (Aranesp®); dasatinib (Sprycel®); daunorubicin liposomal (DanuoXome®); daunorubicin, daunomycin (Daunorubicin®); daunorubicin, daunomycin (Cerubidine®); degarelix (Firmagon®); Denileukin diftitox (Ontak®); dexrazoxane (Zinecard®); dexrazoxane hydrochloride (Totect®); didemnin B; 17-DMAG; docetaxel (Taxotere®); doxorubicin (Adriamycin PFS®); doxorubicin (Adriamycin®, Rubex®); doxorubicin (Adriamycin PFS Injection®); doxorubicin liposomal (Doxil®); dromostanolone propionate (Dromostanolone®); dromostanolone propionate (Masterone Injection®); eculizumab injection (Soliris®); Elliott's B Solution (Elliott's B Solution®); eltrombopag (Promacta®); epirubicin (Ellence®); Epoetin alfa (Epogen®); erlotinib (Tarceva®); estramustine (Emcyt®); ethinyl estradiol; etoposide phosphate (Etopophos®); etoposide, VP-16 (Vepesid®); everolimus tablets (Afinitor®); exemestane (Aromasin®); ferumoxytol (Feraheme Injection®); Filgrastim (Neupogen®); floxuridine (intraarterial) (FUDR®); fludarabine (Fludara®); fluorouracil, 5-FU (Adrucil®); fulvestrant (Faslodex®); gefitinib (Iressa®); geldanamycin; gemcitabine (Gemzar®); gemtuzumab ozogamicin (Mylotarg®); goserelin acetate (Zoladex Implant®); goserelin acetate (Zoladex®); histrelin acetate (Histrelin Implant®); hydroxyurea (Hydrea®); Ibritumomab Tiuxetan (Zevalin®); idarubicin (Idamycin®); ifosfamide (IFEX®); imatinib mesylate (Gleevec®); interferon alfa 2a (Roferon A®); Interferon alfa-2b (Intron A®); iobenguane I 123 injection (AdreView®); irinotecan (Camptosar®); ixabepilone (Ixempra®); lapatinib tablets (Tykerb®); lenalidomide (Revlimid®); letrozole (Femara®); leucovorin (Wellcovorin®, Leucovorin®); Leuprolide Acetate (Eligard®); levamisole (Ergamisol®); lomustine, CCNU (CeeBU®); meclorethamine, nitrogen mustard (Mustargen®); megestrol acetate (Megace®); melphalan, L-PAM (Alkeran®); mercaptopurine, 6-MP (Purinethol®); mesna (Mesnex®); mesna (Mesnex Tabs®); methotrexate (Methotrexate®); methoxsalen (Uvadex®); 8-methoxypsoralen; mitomycin C (Mutamycin®); mitotane (Lysodren®); mitoxantrone (Novantrone®); mitramycin; nandrolone phenpropionate (Durabolin-50®); nelarabine (Arranon®); nilotinib (Tasigna®); Nofetumomab (Verluma®); ofatumumab (Arzerra®); Oprelvekin (Neumega®); oxaliplatin (Eloxatin®); paclitaxel (Paxene®); paclitaxel (Taxol®); paclitaxel protein-bound particles (Abraxane®); palifermin (Kepivance®); pamidronate (Aredia®); panitumumab (Vectibix®); pazopanib tablets (Votrienttm®); pegademase (Adagen (Pegademase Bovine)®); pegaspargase (Oncaspar®); Pegfilgrastim (Neulasta®); pemetrexed disodium (Alimta®); pentostatin (Nipent®); pipobroman (Vercyte®); plerixafor (Mozobil®); plicamycin, mithramycin (Mithracin®); porfimer sodium (Photofrin®); pralatrexate injection (Folotyn®); procarbazine (Matulane®); quinacrine (Atabrine®); rapamycin; Rasburicase (Elitek®); raloxifene hydrochloride (Evista®); Rituximab (Rituxan®); romidepsin (Istodax®); romiplostim (Nplate®); sargramostim (Leukine®); Sargramostim (Prokine®); sorafenib (Nexavar®); streptozocin (Zanosar®); sunitinib maleate (Sutent®); talc (Sclerosol®); tamoxifen (Nolvadex®); temozolomide (Temodar®); temsirolimus (Torisel®); teniposide, VM-26 (Vumon®); testolactone (Teslac®); thioguanine, 6-TG (Thioguanine®); thiopurine; thiotepa (Thioplex®); topotecan (Hycamtin®); toremifene (Fareston®); Tositumomab (Bexxar®); Tositumomab/I-131 tositumomab (Bexxar®); trans-retinoic acid; Trastuzumab (Herceptin®); tretinoin, ATRA (Vesanoid®); triethylenemelamine; Uracil Mustard (Uracil Mustard Capsules®); valrubicin (Valstar®); vinblastine (Velban®); vincristine (Oncovin®); vinorelbine (Navelbine®); vorinostat (Zolinza®); wortmannin; and zoledronate (Zometa®).

In an embodiment of the invention, an anti-CD27 antibody or antigen-binding fragment thereof of the invention (e.g., antibody 131A or a humanized version thereof) is used in association with one or more antiemetics including, but not limited to: casopitant (GlaxoSmithKline), Netupitant (MGI-Helsinn) and other NK-1 receptor antagonists, palonosetron (sold as Aloxi® by MGI Pharma), aprepitant (sold as Emend by Merck and Co.; Rahway, N.J.), diphenhydramine (sold as Benadryl® by Pfizer; New York, N.Y.), hydroxyzine (sold as Atarax® by Pfizer; New York, N.Y.), metoclopramide (sold as Reglan® by AH Robins Co; Richmond, Va.), lorazepam (sold as Ativan® by Wyeth; Madison, N.J.), alprazolam (sold as Xanax® by Pfizer; New York, N.Y.), haloperidol (sold as Haldol® by Ortho-McNeil; Raritan, N.J.), droperidol (Inapsine®), dronabinol (sold as Marinol® by Solvay Pharmaceuticals, Inc.; Marietta, Ga.), dexamethasone (sold as Decadron® by Merck and Co.; Rahway, N.J.), methylprednisolone (sold as Medrol® by Pfizer; New York, N.Y.), prochlorperazine (sold as Compazine by Glaxosmithkline; Research Triangle Park, N.C.), granisetron (sold as Kytril® by Hoffmann-La Roche Inc.; Nutley, N.J.), ondansetron (sold as Zofran® by Glaxosmithkline; Research Triangle Park, N.C.), dolasetron (sold as Anzemet® by Sanofi-Aventis; New York, N.Y.), tropisetron (sold as Navoban® by Novartis; East Hanover, N.J.).

Other side effects of cancer treatment include red and white blood cell deficiency. Accordingly, in an embodiment of the invention, an anti-CD27 antibody or antigen-binding fragment thereof (e.g., antibody 131A or a humanized version thereof) is in association with an agent which treats or prevents such a deficiency, such as, e.g., filgrastim, PEG-filgrastim, erythropoietin, epoetin alfa or darbepoetin alfa.

In an embodiment of the invention, an anti-CD27 antibody or antigen-binding fragment thereof of the invention (e.g., antibody 131A or a humanized version thereof) is administered in association with anti-cancer radiation therapy. For example, in an embodiment of the invention, the radiation therapy is external beam therapy (EBT): a method for delivering a beam of high-energy X-rays to the location of the tumor. The beam is generated outside the patient (e.g., by a linear accelerator) and is targeted at the tumor site. These X-rays can destroy the cancer cells and careful treatment planning allows the surrounding normal tissues to be spared. No radioactive sources are placed inside the patient's body. In an embodiment of the invention, the radiation therapy is proton beam therapy: a type of conformal therapy that bombards the diseased tissue with protons instead of X-rays. In an embodiment of the invention, the radiation therapy is conformal external beam radiation therapy: a procedure that uses advanced technology to tailor the radiation therapy to an individual's body structures. In an embodiment of the invention, the radiation therapy is brachytherapy: the temporary placement of radioactive materials within the body, usually employed to give an extra dose- or boost-of radiation to an area.

In an embodiment of the invention, a surgical procedure is administered in association with an anti-CD27 antibody or antigen-binding fragment thereof (e.g., antibody 131A or a humanized version thereof) is surgical tumorectomy.

In a further embodiment, the patient is infused with autologous T cells expanded ex vivo with anti-CD27 specific antibodies or antigen-binding fragments thereof. In another embodiment, the patient is administered autologous T cells in combination with the anti-CD27 specific antibodies or antigen-binding fragments thereof. In yet another embodiment, the patient is vaccinated with a cancer vaccine, and infused with autologous T cells expanded ex vivo with anti-CD27 specific antibodies or antigen-binding fragments thereof. The autologous T-cells can be autologous infiltrating lymphocytes, T-cells transduced with high affinity T-cell receptors against tumor antigens or T cells transduced with chimeric antigen receptors composed of hybrid immunoglobulin lights chains with endo-domains of T-cell signaling molecules. See Kalos M. and June C. H., Immunity, 39, 2013, p 49-60; Wu R. et al, Cancer J. 2012; 18(2): 160-175; and June, C. H., J. Clin. Invest. 117:1466-1476 (2007).

Experimental and Diagnostic Uses

The anti-CD27 antibodies and antigen-binding fragments thereof disclosed herein (e.g., antibody 131A or a humanized version thereof) may be used as affinity purification agents. In this process, the anti-CD27 antibodies and antigen-binding fragments thereof are immobilized on a solid phase such a Sephadex, glass or agarose resin or filter paper, using methods well known in the art. The immobilized antibody or fragment is contacted with a sample containing the CD27 protein (or a fragment thereof) to be purified, and thereafter the support is washed with a suitable solvent that will remove substantially all the material in the sample except the CD27 protein, which is bound to the immobilized antibody or fragment. Finally, the support is washed with a solvent which elutes the bound CD27 (e.g., protein A). Such immobilized antibodies and fragments form part of the present invention.

Further provided are antigens for generating secondary antibodies which are useful for example for performing Western blots and other immunoassays discussed herein.

Anti-CD27 antibodies (e.g., humanized antibodies) and antigen-binding fragments thereof may also be useful in diagnostic assays for CD27 protein, e.g., detecting its expression in specific cells, tissues, or serum, e.g., tumor cells such as melanoma cells. Such diagnostic methods may be useful in various disease diagnoses.

The present invention includes ELISA assays (enzyme-linked immunosorbent assay) incorporating the use of an anti-CD27 antibody or antigen-binding fragment thereof disclosed herein (e.g., antibody 131A or a humanized version thereof).

For example, such a method comprises the following steps:

(a) coat a substrate (e.g., surface of a microtiter plate well, e.g., a plastic plate) with anti-CD27 antibody or antigen-binding fragment thereof; (b) apply a sample to be tested for the presence of CD27 to the substrate; (c) wash the plate, so that unbound material in the sample is removed; (d) apply detectably labeled antibodies (e.g., enzyme-linked antibodies) which are also specific to the CD27 antigen; (e) wash the substrate, so that the unbound, labeled antibodies are removed; (f) if the labeled antibodies are enzyme linked, apply a chemical which is converted by the enzyme into a fluorescent signal; and (g) detect the presence of the labeled antibody.

Detection of the label associated with the substrate indicates the presence of the CD27 protein.

In a further embodiment, the labeled antibody or antigen-binding fragment thereof is labeled with peroxidase which react with ABTS (e.g., 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid)) or 3,3′,5,5′-Tetramethylbenzidine to produce a color change which is detectable. Alternatively, the labeled antibody or fragment is labeled with a detectable radioisotope (e.g., ³H) which can be detected by scintillation counter in the presence of a scintillant.

An anti-CD27 antibody or antigen-binding fragment thereof of the invention (e.g., antibody 131A or a humanized version thereof) may be used in a Western blot or immune-protein blot procedure. Such a procedure forms part of the present invention and includes e.g.: (1) optionally transferring proteins from a sample to be tested for the presence of CD27 (e.g., from a PAGE or SDS-PAGE electrophoretic separation of the proteins in the sample) onto a membrane or other solid substrate using a method known in the art (e.g., semi-dry blotting or tank blotting); contacting the membrane or other solid substrate to be tested for the presence of bound CD27 or a fragment thereof with an anti-CD27 antibody or antigen-binding fragment thereof of the invention.

Such a membrane may take the form of a nitrocellulose or vinyl-based (e.g., polyvinylidene fluoride (PVDF)) membrane to which the proteins to be tested for the presence of CD27 in a non-denaturing PAGE (polyacrylamide gel electrophoresis) gel or SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) gel have been transferred (e.g., following electrophoretic separation in the gel). Before contacting the membrane with the anti-CD27 antibody or fragment, the membrane is optionally blocked, e.g., with non-fat dry milk or the like so as to bind non-specific protein binding sites on the membrane.

(2) washing the membrane one or more times to remove unbound anti-CD27 antibody or fragment and other unbound substances; and (3) detecting the bound anti-CD27 antibody or fragment.

Detection of the bound antibody or fragment indicates that the CD27 protein is present on the membrane or substrate and in the sample. Detection of the bound antibody or fragment may be by binding the antibody or fragment with a secondary antibody (an anti-immunoglobulin antibody) which is detectably labeled and, then, detecting the presence of the secondary antibody.

The anti-CD27 antibodies and antigen-binding fragments thereof disclosed herein (e.g., antibody 131A and humanized versions thereof) may also be used for immunohistochemistry.

Such a method forms part of the present invention and comprises, e.g.,

(1) contacting a cell (e.g., a tumor cell such as a melanoma cell) to be tested for the presence of CD27 protein with an anti-CD27 antibody or antigen-binding fragment thereof of the invention; and (2) detecting the antibody or fragment on or in the cell.

If the antibody or fragment itself is detectably labeled, it can be detected directly. Alternatively, the antibody or fragment may be bound by a detectably labeled secondary antibody which is detected.

Certain anti-CD27 antibodies and antigen-binding fragments thereof disclosed herein (e.g., antibody 131A and humanized versions thereof) may also be used for in vivo tumor imaging. Such a method may include injection of a radiolabeled anti-CD27 antibody or antigen-binding fragment thereof into the body of a patient to be tested for the presence of a tumor associated with CD27 expression (e.g., which expresses CD27, for example, on the tumor cell surface) followed by nuclear imaging of the body of the patient to detect the presence of the labeled antibody or fragment e.g., at loci comprising a high concentration of the antibody or fragment which are bound to the tumor. The detection of the loci indicates the presence of the CD27⁺ tumor and tumor cells.

Imaging techniques include SPECT imaging (single photon emission computed tomography) or PET imaging (positron emission tomography). Labels include e.g., iodine-123 (¹²³I) and technetium-99m (^(99m)Tc), e.g., in conjunction with SPECT imaging or ¹¹C, ¹³N, ¹⁵O or ¹⁸F, e.g., in conjunction with PET imaging or Indium-111 (See e.g., Gordon et al., (2005) International Rev. Neurobiol. 67:385-440).

Pharmaceutical Compositions and Administration

To prepare pharmaceutical or sterile compositions of the anti-CD27 antibodies and antigen-binding fragments of the invention (e.g., antibody 131A and humanized versions thereof), the antibody or antigen-binding fragment thereof is admixed with a pharmaceutically acceptable carrier or excipient. See, e.g., Remington's Pharmaceutical Sciences and U.S. Pharmacopeia: National Formulary, Mack Publishing Company, Easton, Pa. (1984).

Formulations of therapeutic and diagnostic agents may be prepared by mixing with acceptable carriers, excipients, or stabilizers in the form of, e.g., lyophilized powders, slurries, aqueous solutions or suspensions (see, e.g., Hardman, et al. (2001) Goodman and Gilman's The Pharmacological Basis of Therapeutics, McGraw-Hill, New York, N.Y.; Gennaro (2000) Remington: The Science and Practice of Pharmacy, Lippincott, Williams, and Wilkins, New York, N.Y.; Avis, et al. (eds.) (1993) Pharmaceutical Dosage Forms: Parenteral Medications, Marcel Dekker, NY; Lieberman, et al. (eds.) (1990) Pharmaceutical Dosage Forms: Tablets, Marcel Dekker, NY; Lieberman, et al. (eds.) (1990) Pharmaceutical Dosage Forms: Disperse Systems, Marcel Dekker, NY; Weiner and Kotkoskie (2000) Excipient Toxicity and Safety, Marcel Dekker, Inc., New York, N.Y.).

Toxicity and therapeutic efficacy of the antibodies of the invention, administered alone or in combination with another therapeutic agent, can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD₅₀ (the dose lethal to 50% of the population) and the ED₅₀ (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index (LD₅₀/ED₅₀). The data obtained from these cell culture assays and animal studies can be used in formulating a range of dosage for use in human. The dosage of such compounds lies preferably within a range of circulating concentrations that include the ED₅₀ with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration.

In a further embodiment, a further therapeutic agent that is administered to a subject in association with an anti-CD27 antibody or antigen-binding fragment thereof of the invention (e.g., antibody 131A or humanized versions thereof) in accordance with the Physicians' Desk Reference 2003 (Thomson Healthcare; 57th edition (Nov. 1, 2002)).

The mode of administration can vary. Routes of administration include oral, rectal, transmucosal, intestinal, parenteral; intramuscular, subcutaneous, intradermal, intramedullary, intrathecal, direct intraventricular, intravenous, intraperitoneal, intranasal, intraocular, inhalation, insufflation, topical, cutaneous, transdermal, or intra-arterial.

In particular embodiments, the anti-CD27 antibodies or antigen-binding fragments thereof of the invention (e.g., antibody 131A and humanized versions thereof) can be administered by an invasive route such as by injection. In further embodiments of the invention, an anti-CD27 antibody or antigen-binding fragment thereof, or pharmaceutical composition thereof, is administered intravenously, subcutaneously, intramuscularly, intraarterially, intratumorally, or by inhalation, aerosol delivery. Administration by non-invasive routes (e.g., orally; for example, in a pill, capsule or tablet) is also within the scope of the present invention.

The present invention provides a vessel (e.g., a plastic or glass vial, e.g., with a cap or a chromatography column, hollow bore needle or a syringe cylinder) comprising any of the antibodies or antigen-binding fragments of the invention (e.g., antibody 131A and humanized versions thereof) or a pharmaceutical composition thereof. The present invention also provides an injection device comprising any of the antibodies or antigen-binding fragments of the invention (e.g., antibody 131A and humanized versions thereof) or a pharmaceutical composition thereof. An injection device is a device that introduces a substance into the body of a patient via a parenteral route, e.g., intramuscular, subcutaneous or intravenous. For example, an injection device may be a syringe (e.g., pre-filled with the pharmaceutical composition, such as an auto-injector) which, for example, includes a cylinder or barrel for holding fluid to be injected (e.g., antibody or fragment or a pharmaceutical composition thereof), a needle for piecing skin and/or blood vessels for injection of the fluid; and a plunger for pushing the fluid out of the cylinder and through the needle bore. In an embodiment of the invention, an injection device that comprises an antibody or antigen-binding fragment thereof of the present invention or a pharmaceutical composition thereof is an intravenous (IV) injection device. Such a device includes the antibody or fragment or a pharmaceutical composition thereof in a cannula or trocar/needle which may be attached to a tube which may be attached to a bag or reservoir for holding fluid (e.g., saline; or lactated ringer solution comprising NaCl, sodium lactate, KCl, CaCl₂ and optionally including glucose) introduced into the body of the patient through the cannula or trocar/needle. The antibody or fragment or a pharmaceutical composition thereof may, in an embodiment of the invention, be introduced into the device once the trocar and cannula are inserted into the vein of a subject and the trocar is removed from the inserted cannula. The IV device may, for example, be inserted into a peripheral vein (e.g., in the hand or arm); the superior vena cava or inferior vena cava, or within the right atrium of the heart (e.g., a central IV); or into a subclavian, internal jugular, or a femoral vein and, for example, advanced toward the heart until it reaches the superior vena cava or right atrium (e.g., a central venous line). In an embodiment of the invention, an injection device is an autoinjector; a jet injector or an external infusion pump. A jet injector uses a high-pressure narrow jet of liquid which penetrate the epidermis to introduce the antibody or fragment or a pharmaceutical composition thereof to a patient's body. External infusion pumps are medical devices that deliver the antibody or fragment or a pharmaceutical composition thereof into a patient's body in controlled amounts. External infusion pumps may be powered electrically or mechanically. Different pumps operate in different ways, for example, a syringe pump holds fluid in the reservoir of a syringe, and a moveable piston controls fluid delivery, an elastomeric pump holds fluid in a stretchable balloon reservoir, and pressure from the elastic walls of the balloon drives fluid delivery. In a peristaltic pump, a set of rollers pinches down on a length of flexible tubing, pushing fluid forward. In a multi-channel pump, fluids can be delivered from multiple reservoirs at multiple rates.

The pharmaceutical compositions disclosed herein may also be administered with a needleless hypodermic injection device; such as the devices disclosed in U.S. Pat. Nos. 6,620,135; 6,096,002; 5,399,163; 5,383,851; 5,312,335; 5,064,413; 4,941,880; 4,790,824 or 4,596,556. Such needleless devices comprising the pharmaceutical composition are also part of the present invention. The pharmaceutical compositions disclosed herein may also be administered by infusion. Examples of well-known implants and modules for administering the pharmaceutical compositions include those disclosed in: U.S. Pat. No. 4,487,603, which discloses an implantable micro-infusion pump for dispensing medication at a controlled rate; U.S. Pat. No. 4,447,233, which discloses a medication infusion pump for delivering medication at a precise infusion rate; U.S. Pat. No. 4,447,224, which discloses a variable flow implantable infusion apparatus for continuous drug delivery; U.S. Pat. No. 4,439,196, which discloses an osmotic drug delivery system having multi-chamber compartments. Many other such implants, delivery systems, and modules are well known to those skilled in the art and those comprising the pharmaceutical compositions of the present invention are within the scope of the present invention.

Alternately, one may administer the anti-CD27 antibody or antigen-binding fragment of the invention (e.g., antibody 131A and humanized versions thereof) in a local rather than systemic manner, for example, via injection of the antibody or fragment directly into a tumor, e.g., a CD27⁺ tumor. Furthermore, one may administer the antibody or fragment in a targeted drug delivery system, for example, in a liposome coated with a tissue-specific antibody, targeting, for example, a tumor e.g., a CD27⁺ tumor, e.g., characterized by immunopathology. The liposomes will be targeted to and taken up selectively by the afflicted tissue. Such methods and liposomes are part of the present invention.

The administration regimen depends on several factors, including the serum or tissue turnover rate of the therapeutic antibody or antigen-binding fragment, the level of symptoms, the immunogenicity of the therapeutic antibody, and the accessibility of the target cells in the biological matrix. Preferably, the administration regimen delivers sufficient therapeutic antibody or fragment to effect improvement in the target disease state, while simultaneously minimizing undesired side effects. Accordingly, the amount of biologic delivered depends in part on the particular therapeutic antibody and the severity of the condition being treated. Guidance in selecting appropriate doses of therapeutic antibodies or fragments is available (see, e.g., Wawrzynczak (1996) Antibody Therapy, Bios Scientific Pub. Ltd, Oxfordshire, UK; Kresina (ed.) (1991) Monoclonal Antibodies, Cytokines and Arthritis, Marcel Dekker, New York, N.Y.; Bach (ed.) (1993) Monoclonal Antibodies and Peptide Therapy in Autoimmune Diseases, Marcel Dekker, New York, N.Y.; Baert, et al. (2003) New Engl. J. Med. 348:601-608; Milgrom et al. (1999) New Engl. J. Med. 341:1966-1973; Slamon et al. (2001) New Engl. J. Med. 344:783-792; Beniaminovitz et al. (2000) New Engl. J. Med. 342:613-619; Ghosh et al. (2003) New Engl. J. Med. 348:24-32; Lipsky et al. (2000) New Engl. J. Med. 343:1594-1602).

Determination of the appropriate dose is made by the clinician, e.g., using parameters or factors known or suspected in the art to affect treatment. Generally, the dose begins with an amount somewhat less than the optimum dose and it is increased by small increments thereafter until the desired or optimum effect is achieved relative to any negative side effects. Important diagnostic measures include those of symptoms of, e.g., the inflammation or level of inflammatory cytokines produced. In general, it is desirable that a biologic that will be used is derived from the same species as the animal targeted for treatment, thereby minimizing any immune response to the reagent. In the case of human subjects, for example, humanized and fully human antibodies may be desirable.

Antibodies or antigen-binding fragments thereof disclosed herein (e.g., antibody 131A and humanized versions thereof) may be provided by continuous infusion, or by doses administered, e.g., daily, 1-7 times per week, weekly, bi-weekly, monthly, bimonthly, quarterly, semiannually, annually etc. Doses may be provided, e.g., intravenously, subcutaneously, topically, orally, nasally, rectally, intramuscular, intracerebrally, intraspinally, or by inhalation. A total weekly dose is generally at least 0.05 μg/kg body weight, more generally at least 0.2 μg/kg, 0.5 μg/kg, 1 μg/kg, 10 μg/kg, 100 μg/kg, 0.25 mg/kg, 1.0 mg/kg, 2.0 mg/kg, 5.0 mg/ml, 10 mg/kg, 25 mg/kg, 50 mg/kg or more (see, e.g., Yang, et al. (2003) New Engl. J. Med. 349:427-434; Herold, et al. (2002) New Engl. J. Med. 346:1692-1698; Liu, et al. (1999) J. Neurol. Neurosurg. Psych. 67:451-456; Portielji, et al. (20003) Cancer Immunol. Immunother. 52:151-144). Doses may also be provided to achieve a pre-determined target concentration of anti-CD27 antibody in the subject's serum, such as 0.1, 0.3, 1, 3, 10, 30, 100, 300 μg/ml or more. In other embodiments, an anti-CD27 antibody of the present invention is administered, e.g., subcutaneously or intravenously, on a weekly, biweekly, “every 4 weeks,” monthly, bimonthly, or quarterly basis at 10, 20, 50, 80, 100, 200, 500, 1000 or 2500 mg/subject.

As used herein, the term “effective amount” refer to an amount of an anti-CD27 or antigen-binding fragment thereof of the invention (e.g., antibody 131A and humanized versions thereof) that, when administered alone or in combination with an additional therapeutic agent to a cell, tissue, or subject, is effective to cause a measurable improvement in one or more symptoms of disease, for example cancer or the progression of cancer. An effective dose further refers to that amount of the antibody or fragment sufficient to result in at least partial amelioration of symptoms, e.g., tumor shrinkage or elimination, lack of tumor growth, increased survival time. When applied to an individual active ingredient administered alone, an effective dose refers to that ingredient alone. When applied to a combination, an effective dose refers to combined amounts of the active ingredients that result in the therapeutic effect, whether administered in combination, serially or simultaneously. An effective amount of a therapeutic will result in an improvement of a diagnostic measure or parameter by at least 10%; usually by at least 20%; preferably at least about 30%; more preferably at least 40%, and most preferably by at least 50%. An effective amount can also result in an improvement in a subjective measure in cases where subjective measures are used to assess disease severity.

Kits

Further provided are kits comprising one or more components that include, but are not limited to, an anti-CD27 antibody or antigen-binding fragment, as discussed herein (e.g., antibody 131A or a humanized version thereof) in association with one or more additional components including, but not limited to a pharmaceutically acceptable carrier and/or a therapeutic agent, as discussed herein. The antibody or fragment and/or the therapeutic agent can be formulated as a pure composition or in combination with a pharmaceutically acceptable carrier, in a pharmaceutical composition.

In one embodiment, the kit includes an anti-CD27 antibody or antigen-binding fragment thereof of the invention (e.g., antibody 131A or a humanized version thereof) or a pharmaceutical composition thereof in one container (e.g., in a sterile glass or plastic vial) and/or a therapeutic agent and a pharmaceutical composition thereof in another container (e.g., in a sterile glass or plastic vial).

In another embodiment, the kit comprises a combination of the invention, including an anti-CD27 antibody or antigen-binding fragment thereof of the invention (e.g., humanized 131A) along with a pharmaceutically acceptable carrier, optionally in combination with one or more therapeutic agents formulated together, optionally, in a pharmaceutical composition, in a single, common container.

If the kit includes a pharmaceutical composition for parenteral administration to a subject, the kit can include a device for performing such administration. For example, the kit can include one or more hypodermic needles or other injection devices as discussed above.

The kit can include a package insert including information concerning the pharmaceutical compositions and dosage forms in the kit. Generally, such information aids patients and physicians in using the enclosed pharmaceutical compositions and dosage forms effectively and safely. For example, the following information regarding a combination of the invention may be supplied in the insert: pharmacokinetics, pharmacodynamics, clinical studies, efficacy parameters, indications and usage, contraindications, warnings, precautions, adverse reactions, overdosage, proper dosage and administration, how supplied, proper storage conditions, references, manufacturer/distributor information and patent information.

Detection Kits and Therapeutic Kits

As a matter of convenience, an anti-CD27 antibody or antigen-binding fragment thereof of the invention (e.g., antibody 131A and humanized versions thereof) can be provided in a kit, i.e., a packaged combination of reagents in predetermined amounts with instructions for performing the diagnostic or detection assay. Where the antibody or fragment is labeled with an enzyme, the kit will include substrates and cofactors required by the enzyme (e.g., a substrate precursor which provides the detectable chromophore or fluorophore). In addition, other additives may be included such as stabilizers, buffers (e.g., a block buffer or lysis buffer) and the like. The relative amounts of the various reagents may be varied widely to provide for concentrations in solution of the reagents which substantially optimize the sensitivity of the assay. Particularly, the reagents may be provided as dry powders, usually lyophilized, including excipients which on dissolution will provide a reagent solution having the appropriate concentration.

Also provided are diagnostic or detection reagents and kits comprising one or more such reagents for use in a variety of detection assays, including for example, immunoassays such as ELISA (sandwich-type or competitive format). The kit's components may be pre-attached to a solid support, or may be applied to the surface of a solid support when the kit is used. In some embodiments of the invention, the signal generating means may come pre-associated with an antibody or fragment of the invention or may require combination with one or more components, e.g., buffers, antibody-enzyme conjugates, enzyme substrates, or the like, prior to use. Kits may also include additional reagents, e.g., blocking reagents for reducing nonspecific binding to the solid phase surface, washing reagents, enzyme substrates, and the like. The solid phase surface may be in the form of a tube, a bead, a microtiter plate, a microsphere, or other materials suitable for immobilizing proteins, peptides, or polypeptides. In particular aspects, an enzyme that catalyzes the formation of a chemilluminescent or chromogenic product or the reduction of a chemilluminescent or chromogenic substrate is a component of the signal generating means. Such enzymes are well known in the art. Kits may comprise any of the capture agents and detection reagents described herein. Optionally the kit may also comprise instructions for carrying out the methods of the invention.

Also provided is a kit comprising an anti-CD27 antibody (e.g., humanized antibody) or antigen-binding fragment thereof packaged in a container, such as a vial or bottle, and further comprising a label attached to or packaged with the container, the label describing the contents of the container and providing indications and/or instructions regarding use of the contents of the container to treat one or more disease states as described herein.

In one aspect, the kit is for treating cancer and comprises an anti-CD27 antibody (e.g., humanized antibody) or antigen-binding fragment thereof and a further therapeutic agent or a vaccine. The kit may optionally further include a syringe for parenteral, e.g., intravenous, administration. In another aspect, the kit comprises an anti-CD27 antibody (e.g., humanized antibody) or antigen-binding fragment thereof and a label attached to or packaged with the container describing use of the antibody or fragment with the vaccine or further therapeutic agent. In yet another aspect, the kit comprises the vaccine or further therapeutic agent and a label attached to or packaged with the container describing use of the vaccine or further therapeutic agent with the anti-CD27 antibody or fragment. In certain embodiments, an anti-CD27 antibody and vaccine or further therapeutic agent are in separate vials or are combined together in the same pharmaceutical composition. In addition to the tumor vaccines described above, vaccines for infectious disease may be used in combination with the anti-CD27 antibody or antigen-binding fragment thereof, for example, COMVAX®, M-M-R® II, Pedvax HIB®, PNEUMOVAX® 23, ProQuad®, RotaTeq®, VARIVAX®, and ZOSTAVAX®.

As discussed above in the combination therapy section, concurrent administration of two therapeutic agents does not require that the agents be administered at the same time or by the same route, as long as there is an overlap in the time period during which the agents are exerting their therapeutic effect. Simultaneous or sequential administration is contemplated, as is administration on different days or weeks.

The therapeutic and detection kits disclosed herein may also be prepared that comprise at least one of the antibody, peptide, antigen-binding fragment, or polynucleotide disclosed herein and instructions for using the composition as a detection reagent or therapeutic agent. Containers for use in such kits may typically comprise at least one vial, test tube, flask, bottle, syringe or other suitable container, into which one or more of the detection and/or therapeutic composition(s) may be placed, and preferably suitably aliquoted. Where a second therapeutic agent is also provided, the kit may also contain a second distinct container into which this second detection and/or therapeutic composition may be placed. Alternatively, a plurality of compounds may be prepared in a single pharmaceutical composition, and may be packaged in a single container means, such as a vial, flask, syringe, bottle, or other suitable single container. The kits disclosed herein will also typically include a means for containing the vial(s) in close confinement for commercial sale, such as, e.g., injection or blow-molded plastic containers into which the desired vial(s) are retained. Where a radiolabel, chromogenic, fluorigenic, or other type of detectable label or detecting means is included within the kit, the labeling agent may be provided either in the same container as the detection or therapeutic composition itself, or may alternatively be placed in a second distinct container means into which this second composition may be placed and suitably aliquoted. Alternatively, the detection reagent and the label may be prepared in a single container means, and in most cases, the kit will also typically include a means for containing the vial(s) in close confinement for commercial sale and/or convenient packaging and delivery.

A device or apparatus for carrying out the detection or monitoring methods described herein is also provided. Such an apparatus may include a chamber or tube into which sample can be input, a fluid handling system optionally including valves or pumps to direct flow of the sample through the device, optionally filters to separate plasma or serum from blood, mixing chambers for the addition of capture agents or detection reagents, and optionally a detection device for detecting the amount of detectable label bound to the capture agent immunocomplex. The flow of sample may be passive (e.g., by capillary, hydrostatic, or other forces that do not require further manipulation of the device once sample is applied) or active (e.g., by application of force generated via mechanical pumps, electroosmotic pumps, centrifugal force, or increased air pressure), or by a combination of active and passive forces.

In further embodiments, also provided is a processor, a computer readable memory, and a routine stored on the computer readable memory and adapted to be executed on the processor to perform any of the methods described herein. Examples of suitable computing systems, environments, and/or configurations include personal computers, server computers, hand-held or laptop devices, multiprocessor systems, microprocessor-based systems, set top boxes, programmable consumer electronics, network PCs, minicomputers, mainframe computers, distributed computing environments that include any of the above systems or devices, or any other systems known in the art.

General Methods

Standard methods in molecular biology are described Sambrook, Fritsch and Maniatis (1982 & 1989 2^(nd) Edition, 2001 3^(rd) Edition) Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.; Sambrook and Russell (2001) Molecular Cloning, 3^(rd) ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.; Wu (1993) Recombinant DNA, Vol. 217, Academic Press, San Diego, Calif.). Standard methods also appear in Ausbel, et al. (2001) Current Protocols in Molecular Biology, Vols. 1-4, John Wiley and Sons, Inc. New York, N.Y., which describes cloning in bacterial cells and DNA mutagenesis (Vol. 1), cloning in mammalian cells and yeast (Vol. 2), glycoconjugates and protein expression (Vol. 3), and bioinformatics (Vol. 4).

Methods for protein purification including immunoprecipitation, chromatography, electrophoresis, centrifugation, and crystallization are described (Coligan, et al. (2000) Current Protocols in Protein Science, Vol. 1, John Wiley and Sons, Inc., New York). Chemical analysis, chemical modification, post-translational modification, production of fusion proteins, glycosylation of proteins are described (see, e.g., Coligan, et al. (2000) Current Protocols in Protein Science, Vol. 2, John Wiley and Sons, Inc., New York; Ausubel, et al. (2001) Current Protocols in Molecular Biology, Vol. 3, John Wiley and Sons, Inc., NY, NY, pp. 16.0.5-16.22.17; Sigma-Aldrich, Co. (2001) Products for Life Science Research, St. Louis, Mo.; pp. 45-89; Amersham Pharmacia Biotech (2001) BioDirectory, Piscataway, N.J., pp. 384-391). Production, purification, and fragmentation of polyclonal and monoclonal antibodies are described (Coligan, et al. (2001) Current Protcols in Immunology, Vol. 1, John Wiley and Sons, Inc., New York; Harlow and Lane (1999) Using Antibodies, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.; Harlow and Lane, supra). Standard techniques for characterizing ligand/receptor interactions are available (see, e.g., Coligan, et al. (2001) Current Protocols in Immunology, Vol. 4, John Wiley, Inc., New York).

Monoclonal, polyclonal, and humanized antibodies can be prepared (see, e.g., Sheperd and Dean (eds.) (2000) Monoclonal Antibodies, Oxford Univ. Press, New York, N.Y.; Kontermann and Dubel (eds.) (2001) Antibody Engineering, Springer-Verlag, New York; Harlow and Lane (1988) Antibodies A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., pp. 139-243; Carpenter, et al. (2000) J. Immunol. 165:6205; He, et al. (1998) J. Immunol. 160:1029; Tang et al. (1999) J. Biol. Chem. 274:27371-27378; Baca et al. (1997) J. Biol. Chem. 272:10678-10684; Chothia et al. (1989) Nature 342:877-883; Foote and Winter (1992) J. Mol. Biol. 224:487-499; U.S. Pat. No. 6,329,511).

An alternative to humanization is to use human antibody libraries displayed on phage or human antibody libraries in transgenic mice (Vaughan et al. (1996) Nature Biotechnol. 14:309-314; Barbas (1995) Nature Medicine 1:837-839; Mendez et al. (1997) Nature Genetics 15:146-156; Hoogenboom and Chames (2000) Immunol. Today 21:371-377; Barbas et al. (2001) Phage Display: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.; Kay et al. (1996) Phage Display of Peptides and Proteins: A Laboratory Manual, Academic Press, San Diego, Calif.; de Bruin et al. (1999) Nature Biotechnol. 17:397-399).

Single chain antibodies and diabodies are described (see, e.g., Malecki et al. (2002) Proc. Natl. Acad. Sci. USA 99:213-218; Conrath et al. (2001) J. Biol. Chem. 276:7346-7350; Desmyter et al. (2001) J. Biol. Chem. 276:26285-26290; Hudson and Kortt (1999) J Immunol. Methods 231:177-189; and U.S. Pat. No. 4,946,778). Bifunctional antibodies are provided (see, e.g., Mack, et al. (1995) Proc. Natl. Acad. Sci. USA 92:7021-7025; Carter (2001) J. Immunol. Methods 248:7-15; Volkel, et al. (2001) Protein Engineering 14:815-823; Segal, et al. (2001) J. Immunol. Methods 248:1-6; Brennan, et al. (1985) Science 229:81-83; Raso, et al. (1997) J. Biol. Chem. 272:27623; Morrison (1985) Science 229:1202-1207; Traunecker, et al. (1991) EMBO J. 10:3655-3659; and U.S. Pat. Nos. 5,932,448, 5,532,210, and 6,129,914).

Bispecific antibodies are also provided (see, e.g., Azzoni et al. (1998) J. Immunol. 161:3493; Kita et al. (1999) J. Immunol. 162:6901; Merchant et al. (2000) J. Biol. Chem. 74:9115; Pandey et al. (2000) J. Biol. Chem. 275:38633; Zheng et al. (2001) J. Biol Chem. 276:12999; Propst et al. (2000) J. Immunol. 165:2214; Long (1999) Ann. Rev. Immunol. 17:875).

Purification of antigen is not necessary for the generation of antibodies. Animals can be immunized with cells bearing the antigen of interest. Splenocytes can then be isolated from the immunized animals, and the splenocytes can fused with a myeloma cell line to produce a hybridoma (see, e.g., Meyaard et al. (1997) Immunity 7:283-290; Wright et al. (2000) Immunity 13:233-242; Preston et al., supra; Kaithamana et al. (1999) J. Immunol. 163:5157-5164).

Antibodies can be conjugated, e.g., to small drug molecules, enzymes, liposomes, polyethylene glycol (PEG). Antibodies are useful for therapeutic, diagnostic, kit or other purposes, and include antibodies coupled, e.g., to dyes, radioisotopes, enzymes, or metals, e.g., colloidal gold (see, e.g., Le Doussal et al. (1991) J. Immunol. 146:169-175; Gibellini et al. (1998) J. Immunol. 160:3891-3898; Hsing and Bishop (1999) J. Immunol. 162:2804-2811; Everts et al. (2002) J. Immunol. 168:883-889).

Methods for flow cytometry, including fluorescence activated cell sorting (FACS), are available (see, e.g., Owens, et al. (1994) Flow Cytometry Principles for Clinical Laboratory Practice, John Wiley and Sons, Hoboken, N.J.; Givan (2001) Flow Cytometry, 2^(nd) ed.; Wiley-Liss, Hoboken, N.J.; Shapiro (2003) Practical Flow Cytometry, John Wiley and Sons, Hoboken, N.J.). Fluorescent reagents suitable for modifying nucleic acids, including nucleic acid primers and probes, polypeptides, and antibodies, for use, e.g., as diagnostic reagents, are available (Molecular Probes (2003) Catalogue, Molecular Probes, Inc., Eugene, Oreg.; Sigma-Aldrich (2003) Catalogue, St. Louis, Mo.).

Standard methods of histology of the immune system are described (see, e.g., Muller-Harmelink (ed.) (1986) Human Thymus: Histopathology and Pathology, Springer Verlag, New York, N.Y.; Hiatt, et al. (2000) Color Atlas of Histology, Lippincott, Williams, and Wilkins, Phila, Pa.; Louis, et al. (2002) Basic Histology: Text and Atlas, McGraw-Hill, New York, N.Y.).

Software packages and databases for determining, e.g., antigenic fragments, leader sequences, protein folding, functional domains, glycosylation sites, and sequence alignments, are available (see, e.g., GenBank, Vector NTI® Suite (Informax, Inc, Bethesda, Md.); GCG Wisconsin Package (Accelrys, Inc., San Diego, Calif.); DeCypher® (TimeLogic Corp., Crystal Bay, Nevada); Menne, et al. (2000) Bioinformatics 16: 741-742; Menne, et al. (2000) Bioinformatics Applications Note 16:741-742; Wren, et al. (2002) Comput. Methods Programs Biomed. 68:177-181; von Heijne (1983) Eur. J. Biochem. 133:17-21; von Heijne (1986) Nucleic Acids Res. 14:4683-4690).

Example 1: Immunization and Selection of Anti-CD27 Antibodies

Immunization of Mice with CD27 cDNA

To generate anti-hCD27 antibodies, the cDNA encoding the full length open reading frame of hCD27 was subcloned into the pCI-neo vector (Promega, Madison, Wis.). Expression of the obtained vector was checked by transient transfection of pCI-neo-hCD27 in CHO-K1 cells (American Type Culture Collection, Manassas, Va.) and flow cytometry using 10 μg/ml mouse anti-hCD27 IgG1 (BD Pharmingen #555439), followed by goat anti-mouse IgG1-FITC (1:100) (Southern Biotechnology, Birmingham, Ala.).

Mice were immunized by gene gun immunization using a Helios Gene gun (BioRad, Hercules, Calif.) and DNA coated gold bullets (BioRad) following manufacturer's instructions. Briefly, 1 m gold particles were coated with pCI-neo-hCD27 cDNA and commercial expression vectors for mouse Flt3L and mouse GM-CSF in a 2:1:1 ratio (both from Aldevron, Fargo, N. Dak.). A total of 1 μg of plasmid DNA was used to coat 500 μg of gold particles.

Specifically, 7-8 weeks old female BALB/c mice were immunized in the ears with a gene gun, receiving 3 cycles of a shot in both ears. Approximately, a 1:4,000 anti-hCD27 titer was detected by cell-ELISA in mouse serum after two DNA immunizations. In the cell-ELISA, all incubation steps were followed by a wash step with PBST (PBS with 0.01% Tween 20). Parental CHO-K1 or CHO-K1.hCD27 cells were seeded (40,000 cells/well) in tissue culture plates and incubated overnight at 37° C. The next day, culture medium was removed and cells were incubated for 1 hour with (dilutions of) mouse serum at 37° C. Next, cells were washed with PBST and incubated for 1 hour at 37° C. with 1:1,000 goat-anti-mouse IgG-HRP (Southern Biotechnology, #1030-05).

Subsequently, cells were washed 6 times with PBST and anti-hCD27 immunoreactivity was visualized with 100 μl OptiEIA TMB substrate (BD Biosciences, Franklin Lake, N.J.). Reactions were stopped with 100 μl 0.5 M H2SO₄ and absorbances were read at 460 and 620 nm. Mice that demonstrated reactivity against hCD27 were immunized for a final, fourth time and sacrificed four days later.

Erythrocyte-depleted spleen cell populations were prepared as described previously (Steenbakkers et al., 1992, J. Immunol. Meth. 152: 69-77; Steenbakkers et al., 1994, Mol. Biol. Rep. 19: 125-134) and frozen at −140° C.

Selection of Anti-hCD27 Antibody Producing B Cells

To select B cell clones producing anti-hCD27 antibodies, 1.5×10⁷ erythrocyte-depleted splenocytes were depleted for monocytes. hCD27-specific B-cells were selected by binding on irradiated (3,000 RAD) CHO-K1.hCD27 transfectants at 4° C. or 37° C., which had grown to confluency in a T25-flask. After extensive washing to delete non-specific B-cells, bound B-cells were collected by Trypsin treatment according to the manufacturer's instructions (Invitrogen, cat. no. 25200-056). Next, B-cells were cultured as described by Steenbakkers et al., 1994, Mol. Biol. Rep. 19: 125-134. Briefly, selected B-cells were mixed with 7.5% (v/v) T-cell supernatant and 50,000 irradiated (2,500 RAD) EL-4 B5 nursing cells in a final volume of 200 μl DMEM F12/P/S/10% BCS in a 96-well flat-bottom tissue culture plates.

On day eight, supernatants were screened by cell-ELISA for reactivity to hCD27 as described above. In addition, the hCD27-reactive supernatants were tested for binding to Macaca mulatta CD27 (mmCD27) expressed on CHO-K1 cells. [Macaca mulatta CD27 Genbank Accession No. gi109095214]] In the cell-ELISA, all incubation steps were followed by a wash step with PBST (PBS with 0.01% Tween 20). Parental CHO-K1, CHO-K1.hCD27, or CHO-K1.mmCD27 cells were seeded (40,000 cells/well) in tissue culture plates and incubated overnight at 37° C. The next day, culture medium was removed and cells were incubated for one hour with (dilutions of) B-cell supernatants at 37° C. Next, cells were washed with PBST and incubated for one hour at 37° C. with 1:1,000 goat-anti-mouse IgG-HRP (Southern Biotechnology, #1030-05). Subsequently, cells were washed 6 times with PBST and anti-hCD27 immunoreactivity was visualized with 100 μl TMB Stabilized Chromagen (Invitrogen, cat. no. SB02). Reactions were stopped with 100 μl 0.5 M H2S04 and absorbances were read at 460 and 620 nm.

Subsequently, 64 B-cell clones from the supernatants that showed the binding to hCD27 and mmCD27 were immortalized by mini-electrofusion following published procedures (Steenbakkers et al., 1992, J. Immunol. Meth. 152: 69-77; Steenbakkers et al., 1994, Mol. Biol. Rep. 19:125-34). Specifically, B-cells were mixed with 10⁶ Sp2/0-Ag14 myeloma cells, and serum was removed by washing with DMEM F12 media. Cells were treated with Pronase solution (Calbiochem, cat. no. 4308070.536) for 3 minutes and washed with Electrofusion Isomolar Buffer (Eppendorf, cat. no. 53702). Electrofusions were performed in a 50 μl fusion chamber by an alternating electric field of 30 s, 2 MHz, 400 V/cm followed by a square, high field pulse of 10 is, 3 kV/cm and again by an alternating electric field of 30 s, 2 MHz, 400 V/cm.

Contents of the chamber were transferred to hybridoma selective medium and plated in a 96-well plate under limiting dilution conditions. On day 8 or 9 following the fusions, hybridoma supernatants were screened for hCD27 reactivity, as described above. Hybridoma supernatants were tested in Cell-ELISA experiments for binding to CHO-K1.mmCD27 or CHO-K1 cells expressing hCD27 (A59T), as described above. In addition, cross-competition of the hybridoma supernatants with antibody hCD27.15 was investigated using a Homogeneous Time Resolved Fluorescence (HTRF) assay. Antibody hCD27.15 has been described in WO2012/004367 an antibody produced by a hybridoma deposited with the ATCC having Deposit Accession No. PTA-11008, and having the VH region of SEQ ID NO:3 and a VL region of SEQ ID NO:4 (reference to SEQ ID NOs in WO2012/004367). In this assay 0.6 nM biotinylated hCD27.15 was incubated with 1.2 nM rhCD27-Fc (R&D systems, 382-CD-100), 1.33 nM Streptavidin-K (Acceptor) and 1.25 nM anti-human Fc-D2 (Donor). A serial dilution of the supernatants was added. Cross-competition with hCD27.15 results in reduced energy transfer from donor to acceptor and is expressed as Delta F ((Ratio 665/615 sample-Ratio 665/615 Background)/Ratio 665/615 Background, in which Background is determined by no addition of rhCD27-Fc). Fluorescence was measured on the Victor2 spectrophotometer (PerkinElmer) at 615 and 665 nM.

Finally, hybridoma supernatants were tested for their ability to trigger CD27 signaling. HEK293T cells that were co-transfected with hCD27-pcDNA and a NF-κB-luciferase reporter construct were exposed for 24 hours to serial dilutions of the hybridoma supernatants. Luciferase activity was measured using the Steady Lite Plus High Sensitivity Luminescence Reporter Gene Assay system (PerkinElmer, 6016757) and the Victor spectrophotometer (PerkinElmer).

Next, 46 hybridomas were selected for subcloning by a single round of limiting dilution. After screening limiting dilution supernatants for binding to CHO-K1.hCD27, clones were selected for freezing and storage. Further analysis of the limiting dilution supernatants for binding to CHO-K1.hCD27, cross-competition with hCD27.15, and stimulation of NF-κB, as described above, allowed the selection of 16 hybridomas for serum-free antibody production.

Example 2: Purification and Characterization of Anti-hCD27 Antibodies

Stable hybridomas were cultured in serum-free media for 7 days and supernatants were harvested. Antibodies were purified by mixing with Mab Select SuRe Prot A resin (GE Healthcare 17-5438) and elution from Poly-prep chromatography columns (BioRad 731-1550), according to the manufacturer's instructions. Next, the antibodies were desalted and rebuffered in PBS pH 7.4 (Gibco) using Zeba Spin Desalting Columns (Life Technologies 89889) and quantified using spectrophotometry.

The purified antibodies were subsequently characterized in a series of experiments. As described above, their capability to bind to CHO-K1.hCD27 and CHO-K1.mmCD27 was determined by Cell-ELISA. It was also investigated if they showed cross-competition with hCD27.15 by HTRF assay and if they were able to induce CD27 signaling in the NF-κB luciferase reporter assay. In addition, whether the antibodies had a stimulatory effect on naïve human CD8⁺ T-cells was examined as follows. Untouched naïve CD8⁺ T-cells were isolated from buffy coat using the RosetteSep Human CD8+ T-Cell Enrichment Cocktail (StemCell 15063) and Ficoll gradient centrifugation, followed by MACS-based negative selection using the BD IMag™ human naïve CD8⁺ T-cell enrichment kit (BD cat number 558569), essentially according to the manufacturer's instructions. The isolated CD8⁺ T-cells were checked for purity and naivety by flow cytometry using anti-CD8 and anti-CD45RA antibodies. Next, they were seeded in 96 well-plates at a concentration of 1.5×10⁵ cells/well. Cells were stimulated with soluble anti-CD3 mAb (OKT-3) at a final concentration of 0.125 pg/ml and anti-CD28 mAb (Sanquin, clone 15E8) at a final concentration of 1.0 pig/ml, in presence of serial dilutions of the purified antibodies. After 4 days, the viability of the cells was determined using propidium iodide (PI) and the number of activated cells was determined by flow cytometry.

Based on the results obtained with the above experiments the hCD27.131A antibody was selected for further analysis. Antibody hCD27.131A (or “mouse parental 131A”) is a mouse IgG1antibody having the VH region of SEQ ID NO:7 and a VL region of SEQ ID NO:8.

Example 3: Affinity of Antibody hCD27.15 to hCD27 (A59T) and mmCD27

Previously, agonistic hCD27.15 antibody was isolated as described in WO2012/004367. hCD27.15 was humanized to produce hCD27.15-6B (humanized version of hCD27.15 from WO2012/004367 with identical CDR regions, SEQ ID NOs: 24 and 25) and chimeric hCD27.15-c4 (mouse hCD27.15 variable regions and human IgG4 constant region, SEQ ID NOs: 22 and 23). As shown in FIGS. 1 and 2, respectively, these versions of hCD27.15 antibody do not bind to a frequent occurring SNP in hCD27 (A59T) and do not bind to cynomolgus CD27 (also referred to herein as MmCD27 or Macaca mulatta CD27). In contrast, antibody hCD27.131A was specifically selected to bind to both the frequent occurring SNP in hCD27 (A59T) and cynomolgus CD27.

Example 4: Humanization of hCD27.131A Antibody

The mouse hCD27.131A antibody was humanized using methods described in the specification. From the mouse hCD27.131A antibody, the following humanized variable heavy chains were constructed: 131AVH6, 131AVH7, 131AVH8, 131AVH9 (SEQ ID NOs:10-13); and the following humanized variable light chains were constructed: 131AVL6, 131AVL7, 131AVL8, 131AVL9 (SEQ ID NOs:15-18). Antibodies 131AVH6VL6, 131AVH6VL7, 131AVH6VL8, 131AVH7VL6, 131AVH7VL7, 131AVH7VL8, 131AVH8VL6, 131AVH8VL7, 131AVH8VL8, and 131AVH9VL9 with either human IgG1, IgG2 or IgG4 constant regions were prepared.

Example 5: Binding to Cell-Surface CD27

131AVH6VL6-hIgG1 and 131AVH6VL6-hIgG2 antibodies were expressed in CHO-EXP1 cells or HEK293EXP1 cells. 1F5 hIgG1 (or “1F5”, or “1F5IgG1”, has variable regions of 1F5 in US2011/0274685) was expressed in HEK293EXP1 cells. Purified antibodies were assayed for binding to human CD27 expressing CHOK1 cells, human CD27 A59T expressing CHOK1 cells, and cross-reactivity to rhesus CD27 expressing CHOK1 cells using a cell-based ELISA format. Human CD27 expressing CHOK1 cells, human CD27 A59T expressing CHOK1 cells, and rhesus CD27 expressing CHOK1 cells were plated in 96-well tissue-culture plates in 50 μl of DMEM/F12, 10% BCS and gentamycin (CHO-K1 media). Cells were plated at either 2×10⁴ cells/well two days prior to the assay or 4×10⁴ cells/well one day prior to the assay. Media was removed from the wells prior to the assay followed by incubation of mAb in 100 μL fresh culture medium at a starting concentration of 10 μg/mL and 8 step 1:4 serial dilutions. The antibodies were incubated for 30-60 minutes at room temperature and washed 3 times with PBS/0.05% Tween 20 using a cell ELISA washing protocol on the Biotek EL405x Select CW plate washer. Fifty microliters of the detection antibody (HRP-conjugated goat anti-human IgG (Jackson, cat #109-036-098)), was added at a 1:5000 dilution in CHO-K1 media and incubated at room temperature for 30-60 minutes. Assay plates were washed as above and developed with TMB and stopped with TMB stop solution (KPL cat #50-85-06) or 0.1N phosphoric acid. The plate was read on a Molecular Devices VersaMax plate reader at 450 nm/650 nm. Titration curves were used to determine the half-maximal effective concentration (EC₅₀).

Blood from human healthy volunteers were collected as part of the Palo Alto volunteer blood donor program into tubes containing K2-EDTA (BD vacutainer, BD Biosciences, catalog no. 367863). Blood from rhesus monkeys were collected at Bioreclamation into tubes containing K2-EDTA (BD vacutainer, BD Biosciences, catalog no. 367863), gently inverted, stored at 4° C., and shipped at 4° C. overnight to Merck Research Laboratories, Palo Alto, Calif. Upon receipt, blood was visually confirmed to be devoid of obvious signs of lysis or clotting. One-hundred microliters of blood was incubated in 96-well blocks (Costar, catalog no. 3960) with a cocktail of phenotypic antibodies and the indicated concentrations of test or control antibody for 30 minutes at 4° C. in the dark. Red blood cells (RBCs) were then lysed by incubation with 1.7 mL of Ammonium-Chloride-Potassium red blood cell lysing solution (Life Technologies, catalog no. A10492-01) for 5 minutes. The lysis step was repeated once more with 2 ml of ACK lysing solution, and then again with 300 ml of ACK lysing solution. Cells were then washed after adding 1.7 ml of phosphate buffered saline (PBS) (Hyclone, catalog no. SH30028.02). Cells were then resuspended in 100 μL of PBS containing 0.1 μL Fixable Viability Dye eFluor506 (eBioscience, catalog no. 65-0866-18) and incubated in the dark at 4° C. for 30 minutes. Labeled cells were washed by re-suspending them in 2 mL of staining buffer (SB) containing 2% fetal bovine serum (SAFC, catalog no. 12103C) and 2 mM EDTA (Life technologies, catalog no. 15575-038) in phosphate buffered saline (PBS) (Hyclone, catalog no. SH30028.02) followed by centrifugation at 1300 rpm for 5 minutes. The wash step was repeated with SB as described and then fixed by incubation with 1% paraformaldehyde (Electron Microscopy Sciences, catalog no. 15710) in PBS for 15 minutes at 4° C. in the dark. After a final wash in SB, samples were acquired in a LSR-Fortessa flow cytometer (BD Biosciences) using the high throughput sampler and data analyzed by FlowJo software (Tree Star Inc).

TABLE 3 Binding of 131AVH6VL6-hIgG1 and 1F5-hIgG1 to cell-surface CD27 Flow cytometry MFI cELISA on CD27− on T cells from peripheral blood CHO transfectants CD8+ T cells CD4+ T cells Antibody Target EC50 ± SD (nM) n EC50 ± SD (nM) n EC50 ± SD (nM) n 131AVH6 Human CD27 0.072 ± 0.013 2 0.30 ± 0.07 4 0.32 ± 0.07 4 VL6-hIgG1 Human CD27 A59T 0.097 ± 0.008 2 Not tested Not tested Rhesus CD27 0.054 ± 0.005 2 0.22 ± 0.08 4 0.17 ± 0.07 4 1F5-hIgG1 Human CD27 0.325 ± 0.068 2  1.30 ± 0.595 4  1.54 ± 0.789 4 Human CD27 A59T 0.326 ± 0.049 2 Not tested Not tested Rhesus CD27 0.193 ± 0.063 2 1.92 ± 3.81 4  1.58 ±0.289 4 EC₅₀ = half maximal effective concentration; SD = standard deviation; MFI = mean fluorescence intensity

EC50s for binding to cell surface human CD27 on transfected CHO cells (cELISA) or primary T cells (flow cytometry) were approximately 4-fold lower for 131AVH6VL6-hIgG1 compared to 1F5-hIgG1 (Table 3).

Humanized 131A-hIgG1 and 131A-hIgG4 variant antibodies were expressed in CHO-EXP1 cells or HEK293EXP1 cells. Purified antibodies were assayed for binding to human CD27 expressing CHOK1 cells using a cell-based ELISA format. Human CD27 expressing CHOK1 cells were plated in 96-well tissue-culture plates in 50 μl of DMEM/F12, 10% BCS (bovine calf serum) (CHO-K1 media). Cells were plated at either 2×10⁴ cells/well two days prior to the assay or 4×10⁴ cells/well one day prior to the assay. Media was removed from the wells prior to the assay followed by incubation of mAb in 100 μL fresh culture medium at a starting concentration of 10 μg/mL and 8 step 1:5 serial dilutions (hIgG4 antibodies) or starting concentration of 50 μg/mL and 8 step 1:5 serial dilutions (hIgG1 antibodies). The antibodies were incubated for 30-60 minutes at room temperature and washed 3 times with PBS/0.05% Tween 20 using a cell ELISA washing protocol on the Biotek EL405x Select CW plate washer. Fifty microliters of the detection antibody (HRP-conjugated goat anti-human IgG (Southern Biotech, Cat #2014-05), was added at a 1:2000 dilution in CHO-K1 media and incubated at room temperature for 30-60 minutes. Assay plates were washed as above and developed with TMB and stopped with TMB stop solution (KPL cat #50-85-06) or 0.1N phosphoric acid. The plate was read on a Molecular Devices SpectraMax Plus 384 plate reader at 450 nm/650 nm. Titration curves were used to determine the half-maximal effective concentration (EC₅₀).

TABLE 4 Binding to cell-surface CD27: humanized 131A anti-CD27 antibodies cELISA EC₅₀ (pM) Antibody heavy and light chains hIgG1 hIgG4 131A VH7/VL6 162 42 131A VH7/VL7 207 84 131A VH7/VL8 144 86 131A VH6/VL6 155 101 131A VH6/VL7 130 132 131A VH6/VL8 131 103 131A VH8/VL6 298 97 131A VH8/VL7 262 88 131A VH8/VL8 145 129 131A VH9/VL9 111 130

All humanized 131A-hIgG1 and 131A-hIgG4 variant antibodies had EC50s for binding to cell surface human CD27 on transfected CHO cells (cELISA) that were within 2-fold of the EC50 for 131AVH6VL6-hIgG1 (Table 4), while the EC50 for 1F5-hIgG1 was approximately 4-fold higher compared to 131AVH6VL6-hIgG1 (Table 3).

Blood from human healthy volunteers were obtained as buffy coats from the Stanford blood center and peripheral blood mononuclear cells (PBMCs) were isolated by density centrifugation using Ficoll-Plaque Plus (GE healthcare, #17-1440-03). Remaining red blood cells (RBCs) in PBMC fraction were then lysed by incubation with 2 mL of Ammonium-Chloride-Potassium (ACK) red blood cell lysing solution (Life Technologies, # A10492-01) for 5 minutes at room temperature, and then washed in 10 mL of staining buffer (SB) containing 2% fetal bovine serum (SAFC, #12103C) and 2 mM EDTA (Life technologies, #15575-038) in phosphate buffered saline (PBS) (Hyclone, # SH30028.02) followed by centrifugation at 300 g for 5 minutes. Cells were then counted using Vicell automated cell counter (Beckman Coulter #383556) and 2×10⁵ cells per well were distributed in a U-bottom 96 well plate. Cells were then resuspended in 100 μL of PBS containing 0.1 μL Fixable Viability Dye eFluor506 (eBioscience, #65-0866-18) and incubated in the dark at 4° C. for 30 minutes. Labeled cells were washed by re-suspending them in 150 ul of staining buffer (SB) followed by centrifugation at 300 g for 5 minutes. Cells were then resuspended in 100 ul of SB containing primary anti-CD27 antibodies at various concentrations, incubated in the dark at 4° C. for 30 minutes, followed by washing and centrifugation steps as previously described. Next, cells were stained with a secondary mouse anti-human IgG1 antibody PE conjugated (Southern biotech # HP6001) to detect the humanized anti-CD27 clones, or a Donkey anti-mouse Alexa555 (Thermofisher # A-31570) to detect the parental mouse anti-CD27 antibody. Cells were incubated for 30 minutes at 4° C. in the dark, followed by washing and centrifugation steps. Next, cells were stained with a cocktail of phenotypic antibodies for surface markers (CD3, CD4, CD8, CD11b) and incubated for 30 minutes at 4° C. in the dark, followed by washing and centrifugation steps. Finally, cells were fixed by incubation with 100 ul of BD Cytofix (BD biosciences #554655) for 10 minutes at 4° C. in the dark, followed by washing and centrifugation steps. Samples were acquired in a LSR-Fortessa flow cytometer (BD Biosciences) using the high throughput sampler and data analyzed by FlowJo software (Tree Star Inc).

TABLE 5 Binding of humanized 131A anti-CD27 antibodies to cell-surface CD27 in human T cells EC₅₀ (nM) ± SD EC₅₀ (nM) ± SD Antibody Target CD4+ cells * CD8+ cells * Humanized CD27 131A Human  0.1371 ± 0.00509 0.1234 ± 0.0349 VH6/VL6-hIgG1 CD27 Humanized CD27 131A Human  0.1235 ± 0.00014 0.1097 ± 0.03  VH6/VL7-hIgG1 CD27 Humanized CD27 131A Human 0.12625 ± 0.00714 0.1240 ± 0.018  VH6/VL8-hIgG1 CD27 Humanized CD27 131A Human 0.12815 ± 0.00361 0.1269 ± 0.0314 VH7/VL6-hIgG1 CD27 Humanized CD27 131A Human 0.11345 ± 0.00191 0.1113 ± 0.0249 VH7/VL7-hIgG1 CD27 Humanized CD27 131A Human 0.11825 ± 0.00163 0.1180 ± 0.0130 VH7/VL8-hIgG1 CD27 Humanized CD27 131A Human 0.12445 ± 0.00785 0.1127 ± 0.0203 VH8/VL6-hIgG1 CD27 Humanized CD27 131A Human 0.11475 ± 0.00728 0.1105 ± 0.0178 VH8/VL7-hIgG1 CD27 Humanized CD27 131A Human 0.13465 ± 0.00431 0.1312 ± 0.0293 VH8/VL8-hIgG1 CD27 Humanized CD27 131A Human 0.07736 ± 0.00467 0.0721 ± 0.0174 VH9/VL9-hIgG1 CD27 Mouse Human Chimera Human 0.06644 ± 0.00157 0.0649 ± 0.0186 CD27 131A-hIgG1 CD27 Mouse parental 131A Human 0.06446 ± 0.0102 0.0194 ± 0.0145 CD27 EC₅₀ = half maximal effective concentration; SD = standard deviation; MFI = mean fluorescence intensity * Flow cytometry MFI on T cells from peripheral blood (N = 2 donors)

All humanized 131A-hIgG1 variant antibodies had EC50s for binding to primary T cells (flow cytometry) that were comparable (within 10%) or lower than the EC50 for 131AVH6VL6-hIgG1 (Table 5), while the EC50 for 1F5-hIgG1 was approximately 5-fold higher compared to 131AVH6VL6-hIgG1 (Table 3).

Example 6: Affinity Determination for Binding of Anti-CD27 Antibodies to Human CD27 Recombinant Protein

The kinetic binding activity of anti-human CD27 antibodies 131AVH6VL6-hIgG1 and 131AVH6VL6-hIgG2 was measured by surface plasmon resonance using a Biacore T200 system (Biacore, GE Healthcare, Piscataway, N.J.). Approximately 400 RU of human CD27-Fc fusion protein, approximately 2000 RU of human CD27 A59T-Fc fusion protein or approximately 300 RU of rhesus macaque CD27-Fc fusion protein was immobilized via amine coupling chemistry onto a Series S CM5 sensor chip, catalog number BR-1005-30. HBS-EP+ buffer (BR-1006-69) was used as the running buffer with a flow rate of 50 μL/min. Varying concentrations of 131AVH6VL6-hIgG1 and 131AVH6VL6-hIgG2, ranging from 4.1 nM to 400 nM were injected over the antigen surfaces. Antibody injections lasted 180 seconds and after the injections dissociation was monitored for 900 seconds. Following each injection cycle the antigen surface was regenerated with a 30 second injection of 3M MgCl₂.

Sensograms were “double referenced” by subtracting the response from a blank surface and that from a buffer injection and used for analyzing the rate constant of association (ka) and dissociation (kd), and the equilibrium dissociation constant KD. The resulting data sets were fitted with a 1:1 Langmuir Binding Model using the Biacore T200 evaluation software (version 2.0). Table 6 summarizes the affinities for the anti-human CD27 antibodies to human CD27-Fc fusion protein, human CD27 A59T-Fc fusion protein and rhesus macaque CD27-Fc fusion protein.

TABLE 6 Measurement of Affinity for anti-Human CD27 Antibodies to CD27 Antigen Using BIAcore. Biacore k_(a) Biacore k_(d) Biacore K_(D) Antibody Antigen (M⁻¹s⁻¹) (s⁻¹) (nM) 131AVH6VL6- huCD27 2.2E+05 1.1E−03 5.1 hIgG1 131AVH6VL6- huCD27 2.4E+05 1.5E−03 6.4 hIgG2 131AVH6VL6- huCD27 1.5E+05 1.1E−03 7.3 hIgG1 A59T 131AVH6VL6- huCD27 1.4E+05 1.2E−03 8.2 hIgG2 A59T 131AVH6VL6- rhCD27 2.3E+05 1.0E−03 4.3 hIgG1 131AVH6VL6- rhCD27 2.6E+05 1.4E−03 5.5 hIgG2

Surface plasmon resonance (SPR) experiments were performed using a Biacore 4000 system (Biacore, GE Healthcare, Piscataway, N.J.) to determine the kinetic binding activity of humanized anti-CD27.131A hIgG1 and hIgG4 variants to His9G-tagged human and cynomolgus CD27 recombinant proteins (“His9G” disclosed as SEQ ID NO: 69) (in-house, transient plasmid transfection of HEK293 cells). The surface of a Series S CM5 sensor chip (GE/Biacore, Cat # BR-1005-30) was prepared via amine-coupling of mouse anti-human IgG (Fc) antibody (Human Antibody Capture Kit, GE/Biacore, Cat # BR-1008-39) following the manufacturer's protocol, producing about 9000 RU of immobilized antibody. The assays were performed at 25° C. in filtered and degassed HBS-EP+ running buffer, pH 7.4 (GE/Biacore, Cat #1006-69). The anti-CD27.131A chimeras and variants were captured at 6.6 nM (1 ug/ml) for 120 seconds at a flow rate of 10 uL/minute. Flow cell spots modified with anti-human Fc antibody but lacking CD27 antibodies were used as reference. A five-point 2-fold dilution series (3.13 nM to 50 nM) of His9G-tagged human or cynomolgus CD27 protein (“His9G” disclosed as SEQ ID NO: 69) was injected over the antibody surface for 180 seconds (association phase) at 30 uL/minute followed by 600 seconds of buffer flow (dissociation phase). The chip surface was regenerated with a 30 second injection of 3M MgCl₂ after each injection cycle.

Data were double referenced by subtracting the response from a blank surface and that from a buffer injection and used for analyzing the rate constant of association (ka) and dissociation (kd), and the equilibrium dissociation constant KD. The resulting data sets were fitted with a Langmuir 1:1 binding model using the Biacore 4000 BIAevaluation software, version 1.0 (GE/Biacore). Table 7 summarizes the affinities for the humanized anti-CD27.131A hIgG1 and hIgG4 variants to human and cynomolgus CD27/His proteins.

TABLE 7 Affinity data for the interaction of humanized anti-CD27.131A hIgG1 and hIgG4 variants with His9G-tagged human and cynomolgus CD27 (“His9G” disclosed as SEQ ID NO: 69). KD (M) Humanized anti-CD27 variants Human CD27/His Cyno CD27/His 131A chimera hulgG1 2.92E−08 4.04E−08 hCD27. 131A VH6VL6 hulgG1 6.52E−08 5.15E−08 hCD27. 131A VH6VL7 hulgG1 7.17E−08 4.19E−08 hCD27. 131A VH6VL8 hulgG1 6.82E−08 5.65E−08 hCD27. 131A VH7VL6 hulgG1 4.13E−08 3.48E−08 hCD27. 131A VH7VL7 hulgG1 3.89E−08 3.17E−08 hCD27. 131A VH7VL8 hulgG1 4.49E−08 3.65E−08 hCD27. 131A VH8VL6 hulgG1 6.44E−08 4.66E−08 hCD27. 131A VH8VL7 hulgG1 6.33E−08 4.41E−08 hCD27. 131A VH8VL8 hulgG1 6.10E−08 3.23E−08 hCD27. 131A VH9VL9 hulgG1 6.22E−08 1.05E−07 131A chimera hulgG4 4.34E−08 3.12E−08 hCD27. 131A VH6VL6 hulgG4 1.68E−07 7.30E−08 hCD27. 131A VH6VL7 hulgG4 1.08E−07 8.20E−08 hCD27. 131A VH6VL8 hulgG4 9.91E−08 5.52E−08 hCD27. 131A VH7VL6 hulgG4 6.57E−08 5.82E−08 hCD27. 131A VH7VL7 hulgG4 8.24E−08 3.67E−08 hCD27. 131A VH7VL8 hulgG4 5.85E−08 4.01E−08 hCD27. 131A VH8VL6 hulgG4 1.32E−07 5.97E−08 hCD27. 131A VH8VL7 hulgG4 8.93E−08 7.68E−08 hCD27. 131A VH8VL8 hulgG4 1.10E−07 9.67E−08 hCD27. 131A VH9VL9 hulgG4 1.04E−07 7.29E−08

Example 7: Anti-Tumor Activity of 131AVH6VL6-hIgG1 Compared to 1F5-hIgG1 in a Mouse tumor model

Mice:

B6.Cg-Cd27^(tm1(CD27)Jbo)/Tac mice (huCD27KI mice) were generated by exchanging the extracellular domain of the mouse CD27 gene with the extracellular domain of the human CD27 gene followed by backcrossing to the C56BL6/J background until a 1449 SNP analysis showed 97.95%-98.99% of C57BL/6 recipient genome. Approximately eight to twelve week old female huCD27KI mice with an average body weight of 20.3 grams (range 17.5-23.5 gms) were obtained from a Merck breeding colony maintained at Taconic Laboratory (Germantown, N.Y.). Conventional animal chow and water were provided ad libitum.

Antibody Reagents:

Monoclonal antibodies were obtained from internal sources as frozen (−80° C.) stocks. The 131AVH6VL6-hIgG1 antibody and 1F5-hIgG1 were produced by recombinant cell lines. The mouse IgG2a isotype control (isotype control) was produced from hybridoma cell culture and was specific for infectious bursal disease virus VP2-4-3_GV.

Formulations of Antibody Reagents:

The formulation buffers were specific for each antibody to stabilize proteins and prevent precipitation. The formulations were as follows: isotype control: 20 mM Sodium Acetate, 9% sucrose, pH 5.5; 131AVH6VL6-hIgG1: 20 mM Sodium Acetate, 9% sucrose, pH 5.5; 1F5-hIgG1: 10 mM NaPhosphate+75 mM NaCl+3% Sucrose, pH 7.4.

Tumor Cell Line Preparation and Implant:

MC38 is a cell line derived from a C57BL6/J mouse colon adenocarcinoma. MC38 cells from a frozen stock were maintained in vitro as a monolayer culture in DMEM medium (Cellgro Cat. 10-013CV) supplemented with 10% fetal bovine serum (Hyclone Cat. SH30088.03) at 37° C. in an atmosphere of 5% CO₂ in air. 1×10⁶ log-phase and sub-confluent MC38 cells were injected subcutaneously (SC) in a 100 μL volume of DMEM basal medium in the dorsal right flank of each mouse. Mice were first shaved with electronic clippers in the area that would be used for the implant.

Tumor Measurements and Body Weights:

Tumors were measured the day before the first dose and twice a week thereafter. Tumor length and width were measured using electronic calipers and tumor volume determined using the formula Volume (mm³)=0.5×Length×Width² where length is the longer dimension. Mice were weighed periodically to monitor general health. Before treatment, mice were weighed and tumors from individual mice were measured. To prevent bias, any outliers by weight or tumor volume were removed and the remaining mice distributed into treatment groups with equivalent mean tumor size. When the mean tumor volume in the MC38 tumor-bearing mice reached ˜85 mm³ (range 70-100 mm³), around 5 days post implant, dosing was started. Animals were administered antibodies as described below.

Dosing Solution Preparation, Administration, and Analyses:

Frozen stocks of the antibodies to be tested in the animal model were thawed and transferred to wet ice. To avoid repeated freeze thaw, each vial of stock was thawed once and stored at 4° C. Once thawed the antibodies were used within a month. Before each dosing, stock solution of each antibody was diluted to nominal concentration in the appropriate diluent and dosed immediately. Aliquots of dosing solutions were snap frozen in dry ice and stored at −80° C. until analyses. Dosing solutions were assessed using the Meso Scale Discovery (MSD®, Rockville, Md.) platform which is based on multi-array technology; a combination of electrochemiluminescence detection and patterned arrays.

Dosing and Results:

MC38 tumor-bearing huCD27 knock-in mice were administered 131AVH6V_(L)6-hIgG1, 1F5-hIgG1, or isotype control at a 5 mg/kg dose, IP, every 3-4 days for a total of 7 doses. Post dosing, animals continued to be monitored and tumor volumes were measured twice a week. As demonstrated by the results, which are shown in FIG. 4, the anti-tumor response to 131AVH6VL6-hIgG1 was greater than the anti-tumor response to 1 F5-hIgG1.

Example 8: Anti-Tumor Activity of 131AVH6VL6-hIgG1 in Combination with Anti-PD-1 Antibody in a Mouse Tumor Model

Mice:

Approximately ten to thirteen week old female B6.Cg-Cd27^(tm1(CD27)Jbo)/Tac (huCD27KI) mice with an average body weight of 21.21 grams (range 18.06-23.21 grams) were obtained from a breeding colony maintained at Taconic Laboratory (Germantown, N.Y.). Conventional animal chow and water were provided ad libitum.

Antibody Reagents:

Monoclonal antibodies were obtained from internal sources as frozen (−80° C.) stocks. The 131AVH6VL6-hIgG1 and the anti-murine PD-1 mouse IgG1 antibody (muDX400) were produced by recombinant cell lines. The isotype control was a mouse IgG2a specific for infectious bursal disease virus VP2-4-3_GV and was produced from hybridoma cell culture.

Formulations of Antibody Reagents:

All antibodies were formulated in 20 mM Sodium Acetate, 9% sucrose, pH 5.5 to stabilize the proteins and prevent precipitation.

Tumor Cell Line Preparation and Implant:

MB49 is a tumor cell line derived from a C57BL6/J mouse bladder carcinoma. MB49 cells from a frozen stock were maintained in vitro as a monolayer culture in DMEM medium (Cellgro Cat. 10-013CV) supplemented with 10% fetal bovine serum (Hyclone Cat. SH30088.03) at 37° C. in an atmosphere of 5% CO₂ in air. 5×10⁵ log-phase and sub-confluent MB49 cells were injected subcutaneously (SC) in a 100 μL volume of DMEM basal medium in the dorsal right flank of each mouse. Mice were first shaved with electronic clippers in the area that would be used for the implant.

Tumor Measurements and Body Weights:

Tumors were measured the day before the first dose and twice a week thereafter. Tumor length and width were measured using electronic calipers and tumor volume determined using the formula Volume (mm³)=0.5×Length×Width² where length is the longer dimension. Mice were weighed periodically to monitor general health. Before treatment, mice were weighed and tumors from individual mice were measured. To prevent bias, any outliers by weight or tumor volume were removed and the remaining mice distributed into treatment groups with equivalent mean tumor size. When the mean tumor volume in the MB49 tumor-bearing mice reached ˜91.56 mm³ (range 80.94-102.89 mm³), around 7 days post implant, dosing was started. Animals were administered antibodies as described below.

Dosing Solution Preparation, Administration, and Analyses:

Frozen stocks of the antibodies to be tested in the animal model were thawed and transferred to wet ice. To avoid repeated freeze thaw, each vial of stock was thawed once and aliquots made in volumes sufficient for one time use. Polypropylene, low adhesion tubes were used for this purpose. The aliquots were snap frozen in dry ice and stored at −80° C. Before each dosing, one aliquot was thawed and diluted to nominal concentration in the appropriate diluent and dosed immediately. Aliquots of dosing solutions were snap frozen in dry ice and stored at −80° C. until analyses. Dosing solutions were assessed using the Meso Scale Discovery (MSD®, Rockville, Md.) platform which is based on multi-array technology; a combination of electrochemiluminescence detection and patterned arrays.

Dosing and Results:

MB49 tumor-bearing huCD27 KI mice were administered 131AVH6VL6-hIgG1, muDX400, 131AVH6VL6-hIgG1+muDX400, or isotype control at a 5 mg/kg dose, IP. 131AVH6VL6-hIgG1 was administered every 3-4 days for a total of 6 doses. muDX400 was administered weekly for a total of 4 doses. Post dosing, animals continued to be monitored and tumor volumes were measured twice a week. As demonstrated by the results, which are shown in FIG. 5, there was minimal anti-tumor response to 131AVH6VL6-hIgG1 or muDX400 alone, however combination therapy with 131AVH6VL6-hIgG1+muDX400 led to significantly enhanced anti-tumor efficacy.

Example 9: NF-κB Activation Assay

Cells:

HEK293FT human embryonic kidney cells containing an NF-κB-luciferase reporter construct (pNiFty2-Luc, Invivogen) were previously created by stable transfection methods using Zeocin drug selection (293FT-NF-κB-luciferase cells).

Cell Culture:

293FT-NFfκB-luciferase cells were grown in in DMEM+2 mM Glutamine, (Cellgro) supplemented with 10% Heat Inactivated Fetal Bovine Serum (Hyclone), 1× Pen/strep/glutamine (Cellgro), and 200 ug/mL Zeocin (Life Technologies).

Antibodies:

hCD27.131A IgG1 and IgG4 antibodies were produced by transient expression in either CHO-EXPI or 293-EXPI cells. 1F5 IgG1 was produced by transient expression in 293-EXPI cells. hCD27.15-4B IgG4 was produced by stable expression in CHO cells. All antibodies were purified by standard Protein A methodology.

Transfections: Full-length cDNAs encoding protein for human CD27 WT (Sequence Reference: NP_001233), human CD27 variant p.Ala59Thr (“A59T”, Allele Frequency: 0.2, Sequence Reference: rs25680) or Rhesus/Cynomolgus CD27 (Sequence Reference: XP_001104337.1/XP_005569963.1) were cloned into the pCI-neo expression vector. CD27 plasmids were transiently transfected into 293FT-NFκB-luciferase cells, using Lipofectamine 2000 (Life Technologies). Flow cytometry was used to confirm surface expression of all CD27 constructs.

Stimulations with Antibodies:

Approximately 16-20h after transient transfection of CD27 constructs, cells were harvested with Cell Dissociation buffer (Millipore) or TyrpLE Express (Life), counted and replated in Opti-MEM (Life Technologies) at 5,000-10,000 cells per well into 96-well clear bottom luminescence plates (Corning) and allowed to rest/adhere for 30-180 minutes at 37° C. before stimulation with soluble anti-CD27 antibodies. Soluble anti-hCD27 hCD27.131A IgG1 or hCD27.131A IgG4 (top dose 10 μg/ml), 1F5 IgG1 (top dose 10 μg/ml) and hCD27.15-4B IgG4 (top dose 160 μg/ml) antibodies were then added to cells in triplicate with a 4 fold or 10 fold dilution series, as indicated, cells were incubated for 16-20 hours at 37° C.

Assay for NF-κB Activity:

After incubation with antibodies for 16-20h at 37° C. cells were rested at room temperature for 5 minutes before addition of Bright-Glo (Promega). Plates were then protected from light and incubated for 10 minutes at room temperature before being analyzed on a Luminometer/Luminescence plate reader (Molecular Device-LJL BioSystem or Envision system). Fold change values with antibody were calculated based on conditions with no antibody. Fold change values were used to apply nonlinear curve fits for dose responses using GraphPad Prism software [model: log(agonist) vs. response—variable slope (four parameters) with no constraints applied].

Comparing soluble activity across CD27 isoforms, hCD27.131A VH6VL6 huIgG1 displayed the highest potential for inducing NF-κB activity across all isoforms (FIG. 6). hCD27.131A VH6VL6 huIgG1 showed comparable potency for WT and A59T human CD27 (average EC50 2.22 and 4.72 nM respectively), and approximately 7-fold stronger potency for rhesus CD27 (average 0.29 nM) (Table 8). In contrast, 1F5 huIgG1 was not consistently active in soluble form and hence EC50 values could not be calculated for 1F5 huIgG1 (FIG. 6).

hCD27.15-4B IgG4 (humanized version of hCD27.15 from WO2012/004367 with identical CDR regions, SEQ ID NOs: 41 and 42) consistently showed activity against hCD27 WT expressing cells but not CD27 A59T expressing or Rhesus CD27 expressing cells. This suggests that hCD27.15-4B would not be active against the A59T allele of CD27, present globally at a frequency of 20%. The same trend was seen for the parental hCD27.15 clone (data not shown). Furthermore, the hCD27.15-4B IgG4 mAb is less potent at inducing NF-κB activity vs. hCD27.131AVH6VL6-huIgG1. In these experiments, hCD27.15-4B IgG4 did not induce a plateau for NF-κB signaling even at doses of 160 μg/ml and hence an EC50 value was incalculable. Table 9 shows fold change values for NF-κB activation at a single 2.5 μg/ml concentration to illustrate activity differences at lower doses. For example, at 2.5 μg/ml in WT CD27 expressing cells, hCD27.131A VH6VL6 huIgG1 shows higher potency vs. hCD27.15-4B IgG4 (Ave Fold Change: 2.52±0.45 vs. 1.82±0.38, paired t-test p-value: 0 0.01) or vs. 1F5 huIgG1 (Fold Change: 1.34±0.29, p-value: 0.001). Overall, these data show that hCD27.131A VH6VL6 huIgG1 shows clear NF-κB luciferase activity against WT CD27 and the A59T minor variant allele of CD27 as well as Rhesus CD27 and illustrate how hCD27.131A VH6VL6 huIgG1 shows higher activity vs. 1F5 huIgG1 and more potent activity vs. hCD27.15-4B IgG4.

Several humanized hCD27.131A variants were tested for CD27 induced NF-κB luciferase activity (FIG. 7). All humanized variants showed higher activity than 1F5 IgG1. Direct comparison of chimeric mouse-human hCD27.131A antibodies showed similar activation of chimeric huIgG1 or huIgG4 frameworks on NF-κB luciferase activity (FIG. 7A). Overall, all humanized hCD27.131A huIgG1 variants behaved similarly to each other showing clearly higher NF-κB luciferase activity relative to 1F5 huIgG1 (10 μg/ml median fold change: 5.32 vs. 2.23, FIG. 7A). Similarly, all humanized hCD27.131A IgG4 variants showed higher activity relative to 1F5 huIgG1 (10 μg/ml median fold change: 3.06 vs. 1.73), with a larger activity range observed between individual 131A IgG4 antibodies (FIG. 7B).

TABLE 8 Bioactivity of hCD27.131A VH6VL6 huIgG1, 1F5 huIgG1, hCD27.15-4B IgG4 in an NF-κB-luciferase reporter assay: EC₅₀ and Emax values based on fitted dose response curves. N = number EC₅₀ ± SD Emax ± SD of curve fits Antibody Target (nM) (fold change) determinable hCD27.131A Human CD27 2.22 ± 2.17 2.955 ± 0.362 6 VH6VL6 Human CD27 4.72 ± 1.87 3.220 ± 0.445 5 huIgG1 A59T Rhesus CD27 0.29 ± 0.16 2.111 ± 0.193 5 1F5 huIgG1 Human CD27 NA (Low 1.482 ± 0.266 5 Activity) Human CD27 NA (Low 1.299 ± 0.220 5 A59T Activity) Rhesus CD27 NA (Low 1.333 ± 0.218 3 Activity) hCD27.15-4B Human CD27 NA (No NA (No 0 IgG4 Plateau) Plateau) Human CD27 NA (Low NA (Low 0 A59T Activity) Activity) Rhesus CD27 NA (Low NA (Low 0 Activity) Activity)

TABLE 9 Bioactivity of hCD27.131A VH6VL6 huIgG1, 1F5 huIgG1, hCD27.15-4B IgG4 in an NF-κB-luciferase reporter assay: Fold Change values at 2.5 μg/ml. Fold Change values at Antibody Target 2.5 μg/ml ± SD n hCD27.131A Human CD27 2.52 ± .45 6 VH6VL6 huIgG1 Human CD27 2.61 ± .58 5 A59T Rhesus CD27 2.11 ± .35 5 1F5 huIgG1 Human CD27 1.34 ± .29 6 Human CD27 1.23 ± .22 5 A59T Rhesus CD27 1.22 ± .13 5 hCD27.15-4B Human CD27 1.82 ± .38 3 IgG4 Human CD27 0.97 ± .02 2 A59T Rhesus CD27 0.99 ± .06 2

Example 10: Bioactivity in Primary Human and Rhesus T Cell Co-Stimulation Assays

Human enriched CD8+ T cells were obtained from buffy coats using the RosetteSep Human CD8+ T Cell Enrichment Cocktail (StemCell Technologies, Vancouver, Canada) according to manufacturer's instructions. Briefly, the buffy coat was incubated with the antibody cocktail, diluted with PBS+2% FBS, then centrifuged over a buoyant density medium Ficoll-Paque Plus (GE Healthcare, United Kingdom) to pellet the unwanted cells along with the RBCs. The enriched CD8+ T cells were then removed from the plasma and density medium interface, extensively washed and lysed with ACK lysis buffer (Thermo Fisher, MA, USA). Naïve CD8+ T cells were isolated by further processing the sample using the Human Naïve CD8+ T Cell Enrichment Set (BD Biosciences, CA, USA). Cells were used fresh or either frozen for use in future experiments.

For the activation assay, naïve CD8+ cells were resuspended to 7.5×10⁵ cells/ml in DMEM-F12 (Gibco, CA, USA), 5% heat inactivated human serum (Sigma, MO, USA), 50 μM 2-mercaptoethanol (Sigma, MO, USA), 100 U/ml penicillin and 100 ug/ml streptomycin (Lonza, Switzerland). 1.5×10⁵ cells were cultured in the presence of a sub-optimal dose of αCD3 (0.025-0.05 μg/ml clone OKT3, Biolegend, CA, USA) and αCD28 (1 μg/ml clone 15E8, Cell Sciences, MA, USA) with soluble 131AVH6V_(L)6-hIgG1, 1F5-hIgG1, isotype or anti-CD3/anti-CD28 alone in a flat-bottom 96-well plate for 3 days in a 37 degree Celsius, 5% CO₂ incubator. Following stimulation, cells were washed with PBS, and then stained with a fixable viability dye (eBioscience, CA, USA) for 30 minutes at 4° C. Excess dye was removed by washing and the cells were blocked with TruStain FcX (Biolegend, CA, USA). The cells were then incubated with phenotypic antibodies (FITC mouse anti-human CD69 (clone FN50, BD Biosciences, CA, USA), PE/CF594 mouse anti-human CD4 (clone L200, BD Biosciences, CA, USA), PE/Cy7 mouse anti-human CD25 (clone M-A251, BD Biosciences, CA, USA) and Pacific Blue mouse anti-human CD3 (clone SP34-2, BD Biosciences, CA, USA) and APC/Cy7 mouse anti-human CD8a (clone RPA-T8, Biolegend, CA, USA)) for 30 minutes at 4° C. before being washed and fixed with 1% paraformaldehyde. Activation of T cells was measured by flow cytometry on the BD LSRFortessa™ (BD Biosciences, CA, USA) for surface markers CD25 and CD69. The CD8+ T cell activation is measured by the % CD8+ T cells that are CD25+CD69+.

131AVH6V_(L)6-hIgG1 had activity in the primary CD8+ T cell assay in soluble form (mean 2.3-fold increase relative to isotype control), while 1F5-hIgG1 did not (FIG. 14).

TABLE 10 Fold change in % activated CD8 T cells relative to isotype control donor donor donor donor donor donor donor donor mAb 87 84 91 11 49 56 816 641 Avg SD 131AVH6VL6- 1.55 2.98 1.63 1.96 1.64 0.98 2.43 3.87 2.13 0.93 hIgG1 1F5-hIgG1 1.30 0.96 0.99 1.18 1.05 0.62 0.74 1.13 1.00 0.23

Example 11: Bioactivity in Primary Human Tumor Cultures

Human tumor specimens from patients with non-small cell lung carcinoma (NSCLC), Renal cell carcinoma (RCC), and head & neck carcinoma (H&N) were obtained from commercial vendors in accordance with state and federal regulations.

Digestion of Human Tumor to Generate the Mixed Tumor TIL

Fresh tumor tissues were collected at the operation sites and shipped in AQIX media (AQIX, UK) overnight at 4° C. to Merck Research Laboratories, Palo Alto, Calif. Single cell was dissociated from tumors by fine cutting with a scalpel, followed by a 30-minute incubation at 37° C. in digestion medium containing 10 mL of Dulbecco's Modified Eagle Medium (DMEM) (Cellgro, cat #10-013-CV) with 100 mg/mL collagenase type I (Invitrogen, cat #17100-017), and 10,000 U/mL DNase I (Worthington Biochemical, cat # LS002060). Digested samples were pipetted up and down several times, filtered through a 70-μM strainer, and washed in DMEM complete medium. If the cell viability was less than 30%, Ficoll-density Gradient Separation was performed to enrich live cells. The mixed cells from tumor digestion were applied to the Ficoll-Paque Plus (GE Healthcare, Cat #17-1440-03) density gradient centrifugation at 1000×g for 20 minutes. The enriched live cells were collected from the medium:Ficoll interface and washed 2 times with Dulbecco's phosphate-buffered saline (DPBS).

IFNγ Detection in Tumor TIL Culture Supernatants for Tumor-Infiltrated T Cell Activation

A total 0.1×10⁶/well tumor digested cells were cultured in 96-well round-bottom plates and stimulated with 10 ng/ml soluble anti-CD3 (BioLegend, clone OKT3, cat #371304) in the presence of indicated concentration of 131AVH6VL6-hIgG1 (Transient plasmid transfection of CHO-EXPI cells-suspension culture), anti-PD1 (pembrolizumab), or hCD27.15-4BIgG4, IF5-hIgG1, or isotypes (anti-PCSK9, IgG1 and IgG4). The supernatants were collected at day 6 and IFNγ was measured using the human IFNγ tissue culture kit (MSD, cat # K151AEB).

Since CD27 is expressed on naïve T cells as well as on memory T cells, we hypothesized 131AVH6VL6-hIgG1 could have co-stimulatory effects in tumor infiltrated memory T cells. To evaluate the ability of 131AVH6VL6-hIgG1 to activate T cell in the human tumor immune suppressive environment, we used the mixed cell population from human tumor digestion for the experiments. 131AVH6VL6-hIgG1 0.1, 1, 10, and 20 μg/ml were added to single digested tumor tissue cell culture for 6 days in the presence of 10 ng/ml anti-CD3. IFNγ was measured in the 6 day supernatants. The result from 20 tumors is shown in FIG. 8. 131AVH6VL6-hIgG1 enhanced anti-CD3-induced IFNγ production of human tumor TILs in a dose dependent manner with a statistically significant change at 10 and 20 μg/ml (p<0.001 and p<0.01, respectively). 131AVH6VL6-hIgG1 was also studied by comparing with two other anti-CD27 mAbs, h27.15-4BIgG4 and IF5-IgG1, in the TIL IFNγ production assay from 13 tumors (FIG. 9). 131AVH6VL6-hIgG1 was the only mAb showing a statistically significant increase of anti-CD3-induced IFNγ production (p<0.05).

Example 12: Alanine Scanning for Identification of Epitope of Anti-CD27 Antibodies

The ability of several anti-hCD27 antibodies to bind to a series of hCD27 alanine mutants was determined using CHO-K1 cells, transiently transfected pCI-neo empty vector, pCI-neo.hCD27 and several pCI-neo.CD27Ala mutant constructs. Transfections were carried out in 6 wells plates with 4 tg plasmid DNA and 10 μl Lipofectamine 2000 Reagent (Invitrogen, 11668-019) per well, both diluted in OPTI-MEMI (Gibco, cat. no. 31985), according to the manufacturer's instructions.

After incubating the transfected cells for one day at 37° C. and 5% CO₂ in a humidified incubator, they were washed once in DPBS, detached with 400 μl enzyme-free cell dissociation solution (Gibco, 13151-014) and collected in 800 μl ice-cold MACS buffer (Miltenyi Biotec, 130-091-221). Detached cells were transferred to 96 wells round-bottomed well plates at approximately 1.2×10⁵ cells/well. After spinning down the cells, discarding the supernatants and re-suspending the cells in the residual volume, the primary antibodies were added and incubated for 30 minutes at 4° C. Cells were washed three times with DPBS/BSA 1%, followed by centrifugation, discarding of the supernatants and re-suspension in the residual volume. Binding of the primary antibodies was detected by staining for 30 minutes at 4° C. with goat-anti-mouse IgG-FITC (BD Bioscience, 349031, 2 μl/well) or goat-anti-human IgG-FITC (y-chain specific) (SouthemBiotech, 2040-02, 2 μl/well). After washing once, antibody binding was detected using the HTS plate reader (FACS Canto II) and FlowJo software for data analysis. Debris, dead cells and doublets were excluded from the analysis. Binding was expressed as the geometric mean of the FITC signal, relative to antibody binding to hCD27, which was set at 100%.

As shown in FIG. 10, mutating amino acids P8, H36, R37 and K38 results in loss of binding of mouse hCD27.131A to hCD27, as is indicated by the dark shading. hCD27.131A shows a distinct binding profile from hCD27.15, 1A4, and 1F5IgG1.

Example 13: Epitope Mapping of CD27 and 131AVH6VL6-hIgG1 by X-Ray Crystal Structure Complex Formation and Purification:

CD27/131AVH6VL6Fab complex was formed by incubating at a molar ratio of 2:1 06AOV (CD27) and O1APN (Fab) respectively for 48 hours at 4° C. The reaction mixture was then loaded on Superdex 200 HiLoad 16/600 (120 mL) column and fractions were pooled based on SEC-UPLC analysis. The complex pool was then dialyzed against 25 mM Tris, 100 mM NaCl, pH 8.0. The dialyzed material was centrifuged and filtered over a 22 um syringe filter.

CD27+Fab Crystallization Procedure

Samples for crystallization were prepared by the addition of 6.25 uL of a 40 mM cadmium chloride stock to a 25 uL aliquot of a CD27-Fab complex in a buffer consisting of 25 mM Tris pH=8.0 and 100 mM sodium chloride. The final sample protein concentration was 12.6 mg/ml and the cadmium chloride concentration was 8.0 mM. Samples were held at room temperature for approximately 1 hour prior to setup.

Initial screening was performed using a Topaz free interface diffusion microfluidic system, Topaz 4.96 chips (Fluidigm) and commercially available screens. Chips were set up at room temperature and subsequently held at 18° C. The following conditions were chosen for translation to a vapor diffusion system:

Screen: Rigaku Wizard Cryo 1-2 (Cat #1008649)

Condition: 0.1 M imidazole pH=8.0+40% v/v PEG 400

Screen: Jena Bioscience Classic HTS1 (Cat # CS-201L)

Condition: 0.1 M Tris pH=8.5+30% v/v PEG 3000+0.2 M lithium sulfate

Crystals for X-ray diffraction studies were produced using a sitting drop vapor diffusion method. Plate well conditions consisting of 100 mM Tris pH 8.0-8.5 and PEG 400 30-50% v/v were dispensed using a Formulator (Formulatrix). Drops consisting of equal volumes of well and protein (0.22 uL+0.22 uL) were dispensed at room temperature using an Oryx4 (Douglas Instruments) and subsequently held at 18° C. Crystals grew over a one month period.

Crystals were also produced by the addition of a seed stock prepared in a buffer consisting of 100 mM Tris pH=8.5 and 40% v/v PEG 400. Drops consisting of 0.30 uL protein+0.20 uL well+0.1 uL seed stock were dispensed at room temperature using an Oryx4 (Douglas Instruments) and subsequently held at 18° C. Crystals grew over a one month period.

Crystal Harvesting and Data Collection

A crystal from the crystallization trail #4482 drop F 1 was harvested from the crystallization drop with a 0.3 mm mesh Litholoop (Molecular Dimensions Ltd, Suffold, UK) and cryofrozen in liquid nitrogen. Data collection was performed at the Industrial Macromolecular Crystallography Association (IMCA) beam line, sector 17 of the Advanced Photon Source (APS) at the Argonne National Laboratory (ANL, Lemont, Ill.). Data were collected at a wavelength of 1.0 Å using a Pilatus 6M detector (Dectris AG, Baden-Dattwil, Switzerland). The data were processed using the autoPROC automated processing software with calls to XDS for indexing and integration, AIMLESS for scaling, POINTLESS for data analysis and STARANISO for applying anisotropic diffraction limits. The crystal unit cell parameter is a=114.20, b=126.10, c=131.600, α=90°, β=90°, γ=90°, space group C222(1).

Structure Elucidation and Refinement

The structure was solved by Molecular Replacement using the MOLREP program. PDB entry 2XTJ and 5TLS were used as search probes for the Fab and antigen, respectively. Using default parameters the VH+VL region was located first (Rf/sigma=7.91, TF/sigma=10.83), then the CH1+CL fragment using the variable region as fixed input model for the translation (Rf/sigma=11.56, TF/sigma=23.70). However at this stage the antigen could not be placed unambiguously (best “solution” Rf=4.73, Tf/sigma=3.77). The model was then refined using the autoBUSTER software. Although the values of R_(free) and R_(work) were high (41.4% and 38.4%, respectively), the electron density map showed consistency with most sequence substitutions and insertions or deletions between the Fab used as probe and the final structure. These were corrected using the program COOT. The resulting structure was refined again with autoBUSTER to R_(free) and R_(work) of 34.3% and 30.8%, respectively. Molecular Replacement was attempted again using this model as fixed coordinates, which resulted in an unambiguous solution for the translation function (TF/sigma=17.67). Additional steps of rebuilding and refinement led to final values of R_(free) and R_(work) of 22.4% and 20.0%, respectively. The final model contains CD27 residues 6 to 88 and 94 to 100, Fab light chain residues 1 to 212, Fab heavy chain residues 1 to 131 and 137 to 217, 6 Cadmium cations, one PEG 400 molecule and 288 waters. No ordered glycosylation sites were found.

Analysis of Fab-Antigen Interactions

The following table lists all contacts between Fab and antigen (H-bonds in bold “H”, salt bridges in bold italic (“SB”), a cut-off of 3.30 Å is used for polar interactions):

TABLE 11 List of contacts ≤ 4.0 Å between Antibody and CD27 Fab light chain Antigen Distance (Å) Tyr 31 Oh Pro 8 Ca 3.82 Tyr 31 Oh Pro 8 C 3.81 Tyr 31 Cz Pro 8 Cb 3.74 Tyr 31 Ce2 Pro 8 Cb 3.73 Tyr 31 Ce1 Glu 9 N 3.93 Tyr 31 Oh Glu 9 N  2.95 H Tyr 31 Cz Glu 9 N 3.61 Tyr 31 Oh Glu 9 Ca 3.77 Tyr 31 Oh Glu 9 Cb 3.47 Trp 90 Ch2 His 11 Ce1 3.83 Trp 90 Ch2 His 11 Ne2 3.72 Trp 90 Cz3 His 11 Ne2 3.81 Asp 49 Od2 Arg 37 Cz 3.83 Asp 49 Od1 Arg 37 Cz 3.61 Trp 90 Cz2 Arg 37 Nh1 3.86 Asp 49 Od2 Arg 37 Nh1 3.78 Asp 49 Cg Arg 37 Nh1 3.68 Asp 49 Od1 Arg 37 Nh1

Asp 49 Od2 Arg 37 Nh2

Asp 49 Cg Arg 37 Nh2 3.66 Asp 49 Od1 Arg 37 Nh2 3.53 Tyr 93 Oh Lys 38 Cd 3.47 Tyr 93 Oh Lys 38 Ce 3.53 Tyr 93 Cz Lys 38 Nz 3.98 Tyr 93 Oh Lys 38 Nz  2.93 H Phe 95 Ce2 Lys 38 Nz 3.85 Phe 95 Cz Lys 38 Nz 3.52 Fab heavy chain Antigen Distance (Å) Asn 31 Nd2 Lys 17 Cd 3.60 Asn 31 Nd2 Lys 17 Ce 3.86 Asn 31 Od1 Lys 17 Nz  3.29 H Asn 31 Nd2 Lys 17 Nz 3.87 Asn 31 Cg Lys 17 Nz 3.93 Asn 31 O Leu 18 Cd2 3.32 Asn 31 Cb Leu 18 Cd2 3.82 Thr 30 O Asp 34 O 3.52 Asn 54 Cb Asp 34 O 3.89 Asn 54 Od1 Asp 34 Cb 3.84 Asn 54 Cg Asp 34 Cb 3.93 Asn 52 Nd2 Asp 34 Cg 3.85 Asn 52 Nd2 Asp 34 Od1  3.24 H Asn 52 Nd2 Asp 34 Od2 3.82 Asn 54 Od1 Asp 34 Od2 3.57 Thr 55 Cg2 Asp 34 Od2 3.30 Asn 54 Cb Asp 34 Od2 3.90 Asn 31 O Gln 35 Ca 3.50 Asn 31 O Gln 35 C 3.61 Asn 52 Nd2 Gln 35 Cg 3.43 Asn 52 Od1 Gln 35 Cg 3.40 Asn 52 Cg Gln 35 Cg 3.45 Gly 33 N Gln 35 Cd 3.79 Thr 53 N Gln 35 Cd 3.57 Asn 52 Od1 Gln 35 Cd 3.28 Tyr 32 C Gln 35 Cd 3.94 Thr 53 Og1 Gln 35 Cd 3.64 Asn 52 Cg Gln 35 Cd 3.68 Ile 51 O Gln 35 Oe1 3.98 Gly 33 Ca Gln 35 Oe1 3.12 Gly 33 N Gln 35 Oe1  3.21 H Thr 53 N Gln 35 Oe1  2.76 H Asn 52 Od1 Gln 35 Oe1 3.49 Asn 52 Cg Gln 35 Oe1 3.75 Asn 52 C Gln 35 Oe1 3.43 Thr 53 Ca Gln 35 Oe1 3.79 Thr 53 Cb Gln 35 Oe1 3.94 Tyr 32 C Gln 35 Oe1 3.49 Tyr 32 O Gln 35 Oe1 3.63 Asn 52 Ca Gln 35 Oe1 3.29 Trp 50 Cz3 Gln 35 Oe1 3.81 Thr 53 Og1 Gln 35 Oe1  3.18 H Thr 53 N Gln 35 Ne2 3.90 Asn 52 Od1 Gln 35 Ne2 3.65 Thr 53 Og1 Gln 35 Ne2  3.25 H Tyr 32 N Gln 35 Ne2 3.83 Asn 31 C Gln 35 Ne2 3.76 Asn 54 N Gln 35 Ne2 3.57 Asn 54 Cb Gln 35 Ne2 3.72 Thr 30 O Gln 35 Ne2  2.87 H Thr 30 C Gln 35 Ne2 3.67 Asn 31 O His 36 N  2.88 H Asn 31 O His 36 Ca 3.83 Tyr 32 Cd2 His 36 Cb 3.58 Asn 31 O His 36 Cb 3.62 Tyr 32 Ce2 His 36 Cb 3.66 Tyr 32 Ce2 His 36 Cg 3.72 Asp 101 Od1 His 36 Cd2 3.87 Gly 100 C His 36 Cd2 3.59 Gly 100 Ca His 36 Cd2 3.78 Tyr 32 Ce2 His 36 Cd2 3.71 Tyr 32 Cd2 His 36 Cd2 3.97 Gly 100 O His 36 Cd2 3.44 Asp 101 Od1 His 36 Ce1 3.65 Asp 101 Od1 His 36 Ne2

Asp 101 Cg His 36 Ne2 3.95 Gly 100 O His 36 Ne2 3.79 Gly 100 C His 36 Ne2 3.92 Gly 100 O Arg 37 Cd 3.74 Asp 101 Od1 Arg 37 Ne 3.47 Asp 101 Ca Arg 37 Cz 3.99 Asp 101 Od1 Arg 37 Cz 3.65 Asp 101 Cg Arg 37 Nh2 3.77 Asp 101 Od1 Arg 37 Nh2 3.44 Trp 50 Ch2 Lys 38 Cg 3.93 Trp 50 Ch2 Lys 38 Cd 3.85 Trp 50 Cz2 Lys 38 Cd 3.96 Trp 50 Cz3 Lys 38 Ce 3.80 Trp 50 Ch2 Lys 38 Ce 3.71 Trp 50 Cz2 Lys 38 Ce 3.81 Trp 50 Ce3 Lys 38 Ce 3.99 Trp 50 Ce2 Lys 38 Ce 3.99 Glu 99 Cg Lys 38 Ce 3.89 Glu 99 Cg Lys 38 Nz 3.82

In addition to numerous van der Waals contacts, several H-bonds and salt bridges are involved in the interface. The paratope involves all 3 complementary determining regions (CDR) from each light and heavy chains. The epitope however is limited to the 1^(st) cysteine-rich domain (CRD). In addition to a segment near the N-terminus, residues 8 to 11 and residue 17, all contacts are made with residues 34 to 38 (see FIGS. 11 and 12). The total amount of surface buried upon formation of the antigen-antibody complex is 679A² and 616 A² of CD27 and Fab solvent accessible surfaces, respectively (See FIG. 11).

Example 14: Competition Studies of Anti-CD27 Antibodies for Binding to hCD27

CHO-K1 cells, stably expressing hCD27, were seeded in 96 wells plates (Nunc, 167008) at 4×10⁴ cells/well and incubated at 37° C. and 5% CO₂ in a humidified incubator. Serial dilutions of anti-hCD27 antibodies were allowed to bind for 2 hours at 37° C. and 5% CO₂. Next, a second anti-hCD27 antibody was added at a fixed concentration and incubated for 1 hour at 37° C. and 5% CO₂. The ELx405 BioTEK washer was used to wash the cells three times in DPBS/0.05% Tween. Binding of the anti-hCD27 antibody that was added at a fixed concentration was detected by adding either goat anti-human-IgG-HRP (Jackson Immuno Research, 109-035-088, 1:1000 dilution) or goat anti-mouse-IgG-HRP (Southern Biotech, 1030-05, 1:5000 dilution). Plates were incubated for 1 hour at 37° C. and 5% CO₂ and washed three times, as described above. TMB Stabilized Chromogen (Invitrogen, SB02) was added and cells were incubated for approximately 10 minutes at room temperature. The reaction was stopped by adding an equal volume of 0.5 M H₂SO₄. Finally, the iEMS Reader MF (Labsystems) or the Envision plate reader (Perkin Elmer) was used to measure the absorbance at 460 and 620 nm.

To determine if mouse hCD27.131A and mouse hCD27.15 were able to cross-compete with 1F5IgG1 for binding to hCD27 competition cell-based ELISAs were performed. A serial dilution of 1F5IgG1 was allowed to bind to hCD27 expressed on CHO-K1 cells. Next the binding of mouse hCD27.131A or mouse hCD27.15 was detected. The reverse experiment was also conducted, in which serial dilutions of mouse hCD27.131A or mouse hCD27.15 were allowed to bind, followed by the detection of binding of 1F5IgG1. As is shown in FIG. 13, mouse hCD27.131A does not interfere with the binding of 1F5IgG1, indicating that these two antibodies bind to distinct epitopes on hCD27. Similar data were obtained for mouse hCD27.15, which was shown not to compete with 1F5IgG1 for binding to hCD27. Only when 1F5IgG1 was allowed to bind first, a moderate loss of binding of mouse hCD27.15 could be observed at high concentrations.

This application claims priority to U.S. provisional application No. 62/399,837 and U.S. provisional application No. 62/546,214 incorporated herein by reference in its entirety. All references cited herein are incorporated by reference to the same extent as if each individual publication, database entry (e.g. Genbank sequences or GeneID entries), patent application, or patent, was specifically and individually indicated to be incorporated by reference. This statement of incorporation by reference is intended by Applicants, pursuant to 37 C.F.R. § 1.57(b)(1), to relate to each and every individual publication, database entry (e.g. Genbank sequences or GeneID entries), patent application, or patent, each of which is clearly identified in compliance with 37 C.F.R. § 1.57(b)(2), even if such citation is not immediately adjacent to a dedicated statement of incorporation by reference. The inclusion of dedicated statements of incorporation by reference, if any, within the specification does not in any way weaken this general statement of incorporation by reference. Citation of the references herein is not intended as an admission that the reference is pertinent prior art, nor does it constitute any admission as to the contents or date of these publications or documents. To the extent that the references provide a definition for a claimed term that conflicts with the definitions provided in the instant specification, the definitions provided in the instant specification shall be used to interpret the claimed invention.

The present invention is not to be limited in scope by the specific embodiments described herein. Indeed, various modifications of the invention in addition to those described herein will become apparent to those skilled in the art from the foregoing description and the accompanying figures. Such modifications are intended to fall within the scope of the appended claims.

The foregoing written specification is considered to be sufficient to enable one skilled in the art to practice the invention. Various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description and fall within the scope of the appended claims.

TABLE 12 Sequence Information SEQ ID Description NO: SEQUENCE 131A H - CDR1  1 N

X ₁ N X₁ = M, V, L, I, G, A, S, T 131A H - CDR2  2

X₁ = N or any amino acid except M, C X₂ = T or any amino acid except M, C X₃ = N or any amino acid except M, C X₄ = T or any amino acid except M, C 131A H - CDR3  3

X₁ = M or any amino acid except M, C 131A L - CDR1  4

X₁ = M, V, L, I, G, A, S, T 131A L - CDR2  5

X₁ = D or any amino acid except M, C X₂ = T or any amino acid except M, C 131A L - CDR3  6

X₁ = W or any amino acid except M, C X₂ = N or any amino acid except M, C, P X₃ = S or any amino acid except M, C, P 131A VH  7 QIQLVQSGPELKKPGETVKISCKASGYTFT

WVKQAP PARENTAL GKGLKWMG

RFAFSLETSATTAYLQ INNLKNEDTATYFCAR

WGQGTSVTVSS 131 VL  8 QIVLTQSPAIMSASPGEKVTMTC

WYQQKSGTS PARENTAL PKRWIY

GVPARFSGSGSGTSYSLTISSMEAEDAATY YC

FGSGTKLEIK 131 VH  9 EIQLVQSGAEVKKPGASVKX₁SCKASGYTFT

NWVX₃Q humanized APGQGLKWX₄GX₅

RFTFTLX₁₀TS genus X₁₁X₁₂TAYX₁₃EX₁₄SSLRX₁₅EDTAVYYCAR

WGQ GTTVTVSS X₁ = V, I X₂ = M, V, L, I, G, A, S, T X₃ = K, R X₄ = M, V, L, I X₅ = W or any amino acid except M or C X₆ = N or any amino acid except M, C X₇ = T or any amino acid except M, C X₈ = N or any amino acid except M, C X₉ = T or any amino acid except M, C X₁₀ = D, E X₁₁ = I, A X₁₂ = S, T X₁₃ = M, L, V, I X₁₄ = L, I X₁₅ = S, N X₁₆ = D or any amino acid except M, C X₁₇ = A or any amino acid except M, C X₁₈ = M or any amino acid except C 131A VH6 10 EIQLVQSGAEVKKPGASVKVSCKASGYTFTN

WVKQA (HUMANIZED) PGQGLKWMG

GRFTFTLDTSISTAYM ELSSLRSEDTAVYYCAR

WGQGTTVTVSS 131A VH7 11 EIQLVQSGAEVKKPGASVKISCKASGYTFT

WVKQAP (HUMANIZED) GQGLKWMG

RFTFTLDTSATTAYLE ISSLRSEDTAVYYCAR

WGQGTTVTVSS 131A VH8 12 EIQLVQSGAEVKKPGASVKVSCKASGYTFT

WVKQA (HUMANIZED) PGQGLKWMG

RFTFTLDTSISTAYM ELSSLRNEDTAVYYCAR

WGQGTTVTVSS 131A VH9 13 EIQLVQSGAEVKKPGASVKVSCKASGYTFT

WVRQA (HUMANIZED PGQGLKWMG

RFTFTLDTSASTAYM ELSSLRSEDTAVYYCAR

WGQGTTVTVSS 131A VL 14 X₁IX₂LTQSPX₃TX₄SX₅SX₆GX₇RX₈TX₉X₁₀C

WY humanized QQKPGX₁₂APKRX₁₃IY

GVPARFSGSGSGTX₁₆Y genus X₁₇LTISSX₁₈X₁₉PEDX₂₀AX₂₁YYC

FGQGTKLEI K X₁ = E, D, X₂ = V, Q X₃ = A, S X₄ = L, X₅ = L, A X₆ = P, V X₇ = E, D X₈ = A, V X₉ = L, I X₁₀ = S, T X₁₁ = M, V, L, I, G, A, S, T X₁₂ = Q, K X₁₃ = W or any amino acid except M, C X₁₄ = D or any amino acid except M, C X₁₅ = T or any amino acid except M, C X₁₆ = D, S X₁₇ = S, T X₁₈ = L, M, V, I X₁₉ = E, Q X₂₀ = F, V, L, I, T X₂₁ = V, T X₂₂ = W or any amino acid except M, C X₂₃ = N or any amino acid except M, C, P X₂₄ = S or any amino acid except M, C, P 131A VL6 15 EIVLTQSPATLSLSPGERATLSC

WYQQKPGQA (HUMANIZED) PKRWIY

GVPARFSGSGSGTDYSLTISSLEPEDFAVY YC

FGQGTKLEIK 131A VL7 16 EIVLTQSPATLSLSPGERATLSC

WYQQKPGQA (HUMANIZED) PKRWIY

GVPARFSGSGSGTSYSLTISSLEPEDFATY YC

FGQGTKLEIK 131A VL8 17 EIVLTQSPATLSASPGERVTLSC

WYQQKPGQA (HUMANIZED) PKRWIY

GVPARFSGSGSGTDYSLTISSMEPEDFAVY YC

FGQGTKLEIK 131A VL9 18 DIQLTQSPSTLSASVGDRVTITC

WYQQKPGKA (HUMANIZED) PKRWIY

GVPARFSGSGSGTDYTLTISSLQPEDFATY YC

FGQGTKLEIK Human CD27 19 TPAPKSCPERHYWAQGKLCCQMCEPGTFLVKDCDQHRKAA QCDPCIPGVSFSPDHHTRPHCESCRHCNSGLLVRNCTITANAE CACRNGWQCRDKECTECDPLPNPSLTARSSQALSPHPQPTHL PYVSEMLEARTAGHMQTLADFRQLPARTLSTHWPPQRSLCS SDFIRILVIFSGMFLVFTLAGALFLHQRRKYRSNKGESPVEPA EPCRYSCPREEEGSTIPIQEDYRKPEPACSP Human CD27 20 TPAPKSCPERHYWAQGKLCCQMCEPGTFLVKDCDQHRKTA (A59T) QCDPCIPGVSFSPDHHTRPHCESCRHCNSGLLVRNCTITANAE CACRNGWQCRDKECTECDPLPNPSLTARSSQALSPHPQPTHL PYVSEMLEARTAGHMQTLADFRQLPARTLSTHWPPQRSLCS SDFIRILVIFSGMFLVFTLAGALFLHQRRKYRSNKGESPVEPA EPCRYSCPREEEGSTIPIQEDYRKPEPACSP mmCD27 21 TPAPKSCPERHYWAQGKLCCQMCEPGTFLVKDCDQHRKAA QCHPCIPGVSFSPDHHTRPHCESCRHCNSGLLIRNCTITANAV CACRNGWQCRDKECTECDPPPNPSLTTWPSQALGPHPQPTH LPYVNEMLEARTAGHMQTLADFRHLPARTLSTHWPPQRSLC SSDFIRILVIFSGMFLVFTLAGTLFLHQQRKYRSNKGESPMEP AEPCPYSCPREEEGSTIPIQEDYRKPEPASSP C4-hCD27.15 22 EVRLQQSGADLVKPGASVKLSCTASGFIIKATYMHWVRQRP chimeric EQGLEWIGRIDPANGETKYDPKFQVKATITADTSSSTAYLQL antibody NSLTSDDTAVYYCARYAWYFDVWGAGTTVTVSSASTKGPS heavy chain VFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSG VHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSN TKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMIS RTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREE QFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTI SKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIA VEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQE GNVFSCSVMHEALHNHYTQKSLSLSLGK C4-hCD27.15 23 DIQMTQSPASLSASVGDTVTITCRASENIYSFLAWYHQKQGR chimeric SPQLLVYHAKTLAEGVPSRFSGSGSGTQFSLKINSLQAEDFGS antibody YYCQHYYGSPLTFGAGTKLEVKRTVAAPSVFIFPPSDEQLKS light chain GTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQD SKDSYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFN RGEC hCD27.15 6B 24 QVQLVQSGAEVKKPGASVKVSCKASGFIIKATYMHWVRQA humanized PGQRLEWMGRIDPANGETKYDPKFQVRVTITADTSASTAYM antibody ELSSLRSEDTAVYYCARYAWYFDVWGQGTLVTVSSASTKG heavy chain PSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTS GVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPS NTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMI SRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPRE EQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEK TISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSD IAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRW QEGNVFSCSVMHEALHNHYTQKSLSLSLGK hCD27.15 6B 25 DIQMTQSPSSLSASVGDRVTITCRASENIYSFLAWYQQKPGK humanized APKLLIYHAKTLAEGVTSRFSGSGSGTDFTLTISSLQPEDSAT antibody YYCQHYYGSPLTFGQGTRLEIKRTVAAPSVFIFPPSDEQLKSG light chain TASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDS KDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFN RGEC Leader sequence 26 MEWSWVFLFFLSVTTGVHS heavy chains Leader sequence 27 MSVPTQVLGLLLLWLTDARC light chains Heavy chain 28 ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWN constant SGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCN domain- VDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKP IgG4 KDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNA S228P KTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKG LPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVK GFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTV DKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK Kappa light 29 RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWK chain constant VDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHK domain VYACEVTHQGLSSPVTKSFNRGEC Heavy chain 30 ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWN constant SGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNV domain- NHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFP IgG1 PKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVH NAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSN KALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCL VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKL TVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Heavy chain 31 ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWN constant domain- SGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCN IgG2 VDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKP KDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNA KTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKG LPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVK GFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTV DKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 131 VH genus1 32 Y₁IQLVQSGY₂EY₃KKPGY₄Y₅VKX₁SCKASGYTFT

WV X₃QAPGY₆GLKWX₄G

RFY₇FY₈L X₁₀TSX₁₁X₁₂TAYX₁₃EX₁₄SSLRX₁₅EDTAVYYCAR

DY WGQGTY₉VTVSS Y₁ = E or Q Y₂ = A or P Y₃ = V or L Y₄ = A or E Y₅ = S or T Y₆ = Q or K Y₇ = A or T Y₈ = T or S Y₉ = T or S X₁ = V, I X₂ = M, V, L, I, G, A, S, T X₃ = K, R X₄ = M, V, L, I X₅ = W or any amino acid except M or C X₆ = N or any amino acid except M, C X₇ = T or any amino acid except M, C X₈ = N or any amino acid except M, C X₉ = T or any amino acid except M, C X₁₀ = D, E X₁₁ = I, A X₁₂ = S, T X₁₃ = M, L, V, I X₁₄ = L, I X₁₅ = S, N X₁₆ = D or any amino acid except M, C X₁₇ = A or any amino acid except M, C X₁₈ = M or any amino acid except C 131 VL genus1 33 X₁IX₂LTQSPX₃Y₁X₄SX₅SX₆GX₇Y₂X₈TX₉X₁₀C

W YQQKY₃GX₁₂Y4PKRX₁₃IY

GVPARFSGSGSGTX₁₆ YX₁₇LTISSX₁₈X₁₉Y₅EDX₂₀AX₂₁YYC

FGQGT KLEIK Y₁ = T or I Y₂ = K or R Y₃ = P or S Y₄ = A or S Y₅ = A or P X₁ = E, D, Q X₂ = V, Q X₃ = A, S X₄ = L, M X₅ = L, A X₆ = P, V X₇ = E, D X₈ = A, V X₉ = L, I, M X₁₀ = S, T X₁₁ = M, V, L, I, G, A, S, T X₁₂ = Q, K, T X₁₃ = W or any amino acid except M, C X₁₄ = D or any amino acid except M, C X₁₅ = T or any amino acid except M, C X₁₆ = D, S X₁₇ = S, T X₁₈ = L, M, V, I X₁₉ = E, Q X₂₀ = F, V, L, I, T, A X₂₁ = V, T X₂₂ = W or any amino acid except M, C X₂₃ = N or any amino acid except M, C, P X₂₄ = S or any amino acid except M, C, P 131 VH genus2 34 Y₁IQLVQSGY₂EY₃KKPGY₄Y₅VKX₁SCKASGYTFT

WV X₃QAPGY₆GLY₇WX₄G

RFY₈FY₉L X₁₀TSX₁₁X₁₂TAYX₁₃EX₁₄SSLRX₁₅EDTAVYYCAR

WGQGTY₁₀VTVSS Y₁ = E or Q Y₂ = A or P Y₃ = V or L Y₄ = A or E Y₅ = S or T Y₆ = Q or K Y₇ = K or E Y₈ = A or T Y₉ = T or S Y₁₀ = T or S X₁ = V, I X₂ = M, V, L, I, G, A, S, T X₃ = K, R X₄ = M, V, L, I X₅ = W, F, or Y X₆ = N or Q X₇ = T X₈ = N or Q X₉ = T X₁₀ = D, E X₁₁ = I, A X₁₂ = S, T X₁₃ = M, L, V, I X₁₄ = L, I X₁₅ = S, N X₁₆ = D X₁₇ = A X₁₈ = M, L, V, I 131 VL genus2 35 X₁IX₂LTQSPX₃Y₁X₄SX₅SX₆GX₇Y₂X₈TX₉X₁₀C

W YQQKY₃GX₁₂Y₄PKY₅X₁₃IY

GVPARFSGSGSGTX₁₆ YX₁₇LTISSX₁₈X₁₉Y₆EDX₂₀AX₂₁YYC

FGQGT KLEIK Y₁ = T or I Y₂ = K or R Y₃ = P or S Y₄ = A or S Y₅ = R or L Y₆ = A or P X₁ = E, D, Q X₂ = V, Q X₃ = A, S X₄ = L, M X₅ = L, A X₆ = P, V X₇ = E, D X₈ = A, V X₉ = L, I, M X₁₀ = S, T X₁₁ = M, V, L, I, G, A, S, T X₁₂ = Q, K, T X₁₃ = W or L X₁₄ = D X₁₅ = T X₁₆ = D, S X₁₇ = S, T X₁₈ = L, M, V, I X₁₉ = E, Q X₂₀ = F, V, L, I, T, A X₂₁ = V, T X₂₂ = W, F, or Y X₂₃ = N or Q X₂₄ = S 131A6 36 EIVLTQSPATLSLSPGERATLSCSASSSVSYMHWYQQKPGQA (humanized) PKRWIYDTSKLASGVPARFSGSGSGTDYSLTISSLEPEDFAVY light chain YCQQWNSYPFTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGT ASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSK DSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNR GEC 131A6 IgG1 37 EIQLVQSGAEVKKPGASVKVSCKASGYTFTNYGMNWVKQA (humanized) PGQGLKWMGWINTNTGEPTYAEEFKGRFTFTLDTSISTAYM heavy chain ELSSLRSEDTAVYYCAREGDAMDYWGQGTTVTVSSASTKG PSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALT SGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPS NTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKD TLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKT KPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALP APIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGF YPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDK SRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 131A6 IgG2 38 EIQLVQSGAEVKKPGASVKVSCKASGYTFTNYGMNWVKQA (humanized) PGQGLKWMGWINTNTGEPTYAEEFKGRFTFTLDTSISTAYM heavy chain ELSSLRSEDTAVYYCAREGDAMDYWGQGTTVTVSSASTKG PSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTS GVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPS NTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMI SRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPRE EQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEK TISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDI AVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQ QGNVFSCSVMHEALHNHYTQKSLSLSPGK 131 VH 39 EIQLVQSGAEVKKPGASVKX₁SCKASGYTFT

WVX₃Q humanized APGQGLKWX₄G

RFTFTLX₁₀TS consensus X₁₁X₁₂TAYX₁₃EX₁₄SSLRX₁₅EDTAVYYCAR

WGQ GTTVTVSS X₁ = V, I X₂ = M X₃ = K, R X₄ = M X₅ = W X₆ = N X₇ = T X₈ = N X₉ = T X₁₀ = D X₁₁ = I, A X₁₂ = S, T X₁₃ = M, L X₁₄ = L, I X₁₅ = S X₁₆ = D X₁₇ = A X₁₈ = M 131 VL 40 X₁IX₂LTQSPX₃TX₄SX₅SX₆GX₇RX₈TX₉X₁₀C

WY humanized QQKPGX₁₂APKRX₁₃IY

GVPARFSGSGSGTX₁₆Y consensus X₁₇LTISSX₁₈X₁₉PEDX₂₀AX₂₁YYC

FGQGTKLEI K X₁ = E, D, X₂ = V, Q X₃ = A, S X₄ = L, X₅ = L, A X₆ = P, V X₇ = E, D X₈ = A, V X₉ = L, I X₁₀ = S, T X₁₁ = M X₁₂ = Q, K X₁₃ = W X₁₄ = D X₁₅ = T X₁₆ = D, S X₁₇ = S, T X₁₈ = L, M X₁₉ = E, Q X₂₀ = F X₂₁ = V, T X₂₂ = W X₂₃ = N X₂₄ = S hCD27.15 4B 41 QVQLVQSGAEVKKPGASVKVSCKASGFIIKATYMHWVRQA heavy chain PGQRLEWMGRIDPANGETKYDPKFQVRVTITADTSASTAYM ELSSLRSEDTAVYYCARYAWYFDVWGQGTLVTVSSASTKG PSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTS GVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPS NTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMI SRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPRE EQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEK TISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSD IAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRW QEGNVFSCSVMHEALHNHYTQKSLSLSLGK hCD27.15 4B 42 DIQMTQSPSSLSASVGDRVTITCRASENIYSFLAWYQQKPGK light chain APKLLIYHAKTLAEGVPSRFSGSGSGTDFTLTISSLQPEDFAT YYCQHYYGSPLTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKS GTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQD SKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSF NRGEC CD27 (06AOV) 43 TPAPKSCPERHYWAQGKLCCQMCEPGTFLVKDCDQHRKAA QCDPCIPGVSFSPDHHTRPHCESCRHCNSGLLVRNCTITANAE CACRNGWQCRDKECTECDPLPNPSLTARSSQALSPHPQPTHL PYVSEMLEARTAGHMQTLADFRQLPARTLSTHHHHHHHH 131AVH6VL6 44 EIQLVQSGAEVKKPGASVKVSCKASGYTFTNYGMNWVKQA Fab heavy chain PGQGLKWMGWINTNTGEPTYAEEFKGRFTFTLDTSISTAYM ELSSLRSEDTAVYYCAREGDAMDYWGQGTTVTVSSASTKG PSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALT SGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPS NTKVDKKVEPKSCDKTHT 131AVH6VL6 45 EIVLTQSPATLSLSPGERATLSCSASSSVSYMHWYQQKPGQA Fab light chain PKRWIYDTSKLASGVPARFSGSGSGTDYSLTISSLEPEDFAVY YCQQWNSYPFTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGT ASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSK DSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNR GEC DNA sequence 46 ATGGACATGCGGGTGCCAGCTCAGCTGCTGGGCCTGCTGC encoding TGCTGTGGCTGAGAGGCGCCAGATGCGAGATCGTGCTGAC 131AVH6VL6 CCAGTCCCCCGCCACCCTGTCTCTGAGCCCTGGCGAGAGA light chain GCCACCCTGAGCTGCTCCGCCTCCTCCTCCGTGTCCTACAT (signal peptide GCACTGGTATCAGCAGAAGCCCGGCCAGGCCCCCAAGCG underlined) GTGGATCTACGACACCTCCAAGCTGGCCTCCGGCGTGCCC GCCAGATTCTCCGGCTCTGGCTCTGGCACCGACTACTCCC TGACCATCTCCAGCCTGGAACCCGAGGACTTCGCCGTGTA CTACTGCCAGCAGTGGAACTCCTACCCCTTCACCTTCGGC CAGGGCACCAAGCTGGAAATCAAGCGTACGGTGGCTGCA CCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAA ATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCT ATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACG CCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCA GGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCT GACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTA CGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTC ACAAAGAGCTTCAACAGGGGAGAGTGTTGA(stop codon) DNA sequence 47 ATGGGCTCCACCGCCATCCTGGGACTGCTGCTGGCTGTGC encoding TGCAGGGCGTGTGCGCCGAGATCCAGCTGGTGCAGTCTGG 131AVH6VL6 CGCCGAAGTGAAGAAACCTGGCGCCTCCGTGAAGGTGTC heavy chain CTGCAAGGCCTCCGGCTACACCTTCACCAACTACGGCATG (signal peptide AACTGGGTGAAACAGGCCCCAGGCCAGGGCCTGAAGTGG underlined) ATGGGCTGGATCAACACCAACACCGGCGAGCCCACCTAC GCCGAAGAGTTCAAGGGCCGGTTCACCTTCACCCTGGACA CCTCCATCTCCACCGCCTACATGGAACTGTCCTCCCTGCG GAGCGAGGACACCGCCGTGTACTACTGCGCCCGAGAGGG CGACGCCATGGACTATTGGGGCCAGGGCACAACCGTGAC CGTGTCCTCCGCTAGCACCAAGGGCCCATCGGTCTTCCCC CTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGG CCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGT GACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGT GCACACCTTCCCGGCCGTCCTACAGTCCTCAGGACTCTAC TCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGG GCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAG CAACACCAAGGTGGACAAGAAGGTTGAGCCCAAATCTTG TGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAA CTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAAC CCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCAC ATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGT CAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAAT GCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACG TACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACT GGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACA AAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGC CAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCC CCCATCCCGGGACGAGCTGACCAAGAACCAGGTCAGCCT GACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCC GTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTAC AAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCT TCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGC AGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGC TCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCT CCGGGTAAATGA(stop codon) VH1-102 64 QVQLVQSGAEVKKPGASVKVSCKASGYTFTWVRQAPGQGL EWMGRVTMTRDTSISTAYMELSRLRSDDTAVYYCAR VH1-146 65 QVQLVQSGAEVKKPGASVKVSCKASGYTFTWVRQAPGQGL EWMGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCAR VK1-O2 66 DIQMTQSPSSLSASVGDRVTITCWYQQKPGKAPKLLIYGVPS RFSGSGSGTDFTLTISSLQPEDFATYYC VK3-L6 67 EIVLTQSPATLSLSPGERATLSCWYQQKPGQAPRLLIYGIPAR FSGSGSGTDFTLTISSLEPEDFAVYYC

TABLE 13 Exemplary PD-1 Antibody Sequences SEQ Antibody ID Feature Amino Acid Sequence NO. Pembrolizumab Light Chain CDR1 RASKGVSTSGYSYLH 75 CDR2 LASYLES 76 CDR3 QHSRDLPLT 77 Variable EIVLTQSPATLSLSPGERATLSCRASKGVSTSGYSYLHWY 78 Region QQKPGQAPRLLIYLASYLESGVPARFSGSGSGTDFTLTIS SLEPEDFAVYYCQHSRDLPLTFGGGTKVEIK Light  EIVLTQSPATLSLSPGERATLSCRASKGVSTSGYSYLHWY 48 Chain QQKPGQAPRLLIYLASYLESGVPARFSGSGSGTDFTLTIS SLEPEDFAVYYCQHSRDLPLTFGGGTKVEIKRTVAAPSVF IFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQ SGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYAC EVTHQGLSSPVTKSFNRGEC Pembrolizumab Heavy Chain CDR1 NYYMY 49 CDR2 GINPSNGGTNFNEKFKN 50 CDR3 RDYRFDMGFDY 51 Variable QVQLVQSGVEVKKPGASVKVSCKASGYTFTNYYMYWV 52 Region RQAPGQGLEWMGGINPSNGGTNFNEKFKNRVTLTTDSST TTAYMELKSLQFDDTAVYYCARRDYRFDMGFDYWGQG TTVTVSS Heavy QVQLVQSGVEVKKPGASVKVSCKASGYTFTNYYMYWV 53 Chain RQAPGQGLEWMGGINPSNGGTNFNEKFKNRVTLTTDSST TTAYMELKSLQFDDTAVYYCARRDYRFDMGFDYWGQG TTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYF PEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPS SSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAP EFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPE VQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLH QDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVY TLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPE NNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSV MHEALHNHYTQKSLSLSLGK Nivolumab Light Chain CDR1 RASQSVSSYLA 54 CDR2 DASNRAT 55 CDR3 QQSSNWPRT 56 Variable EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKP 57 Region GQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEP EDFAVYYCQQSSNWPRTFGQGTKVEIK Light  EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKP 58 Chain GQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEP EDFAVYYCQQSSNWPRTFGQGTKVEIKRTVAAPSVFIFPP SDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNS QESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTH QGLSSPVTKSFNRGEC Nivolumab Heavy Chain CDR1 NSGMH 59 CDR2 VIWYDGSKRYYADSVKG 60 CDR3 NDDY 61 Variable QVQLVESGGGVVQPGRSLRLDCKASGITFSNSGMHWVR 62 Region QAPGKGLEWVAVIWYDGSKRYYADSVKGRFTISRDNSK NTLFLQMNSLRAEDTAVYYCATNDDYWGQGTLVTVSS Heavy QVQLVESGGGVVQPGRSLRLDCKASGITFSNSGMHWVR 63 Chain QAPGKGLEWVAVIWYDGSKRYYADSVKGRFTISRDNSK NTLFLQMNSLRAEDTAVYYCATNDDYWGQGTLVTVSSA STKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSW NSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTY TCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVF LFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYV DGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKE YKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEM TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPV LDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNH YTQKSLSLSLGK

TABLE 14 Structure coordinates from crystal structure of 131AVH6VL6Fab and CD27 complex (Table discloses SEQ ID Nos: 70-74, respectively, in order of appearance) ATOM 1 O SER A 6 −8.803 −39.519 13.045 1.00 82.60 O ATOM 2 N SER A 6 −5.820 −40.853 12.360 1.00 85.52 N ATOM 3 CA SER A 6 −7.246 −41.157 12.225 1.00 83.74 C ATOM 4 C SER A 6 −8.079 −40.476 13.335 1.00 80.98 C ATOM 5 CB SER A 6 −7.470 −42.668 12.227 1.00 86.62 C ATOM 6 OG SER A 6 −7.019 −43.264 13.431 1.00 95.24 O ATOM 7 N CYS A 7 −7.966 −40.963 14.593 1.00 69.90 N ATOM 8 CA CYS A 7 −8.687 −40.428 15.760 1.00 65.48 C ATOM 9 C CYS A 7 −7.716 −39.655 16.652 1.00 67.51 C ATOM 10 O CYS A 7 −6.507 −39.865 16.540 1.00 67.71 O ATOM 11 CB CYS A 7 −9.350 −41.557 16.550 1.00 61.81 C ATOM 12 SG CYS A 7 −10.673 −42.420 15.667 1.00 65.96 S ATOM 13 N PRO A 8 −8.230 −38.834 17.599 1.00 62.53 N ATOM 14 CA PRO A 8 −7.327 −38.139 18.528 1.00 62.23 C ATOM 15 C PRO A 8 −6.703 −39.122 19.538 1.00 62.80 C ATOM 16 O PRO A 8 −6.993 −40.316 19.500 1.00 59.20 O ATOM 17 CB PRO A 8 −8.219 −37.078 19.197 1.00 63.97 C ATOM 18 CG PRO A 8 −9.601 −37.363 18.767 1.00 67.86 C ATOM 19 CD PRO A 8 −9.537 −38.155 17.515 1.00 64.20 C ATOM 20 N GLU A 9 −5.835 −38.624 20.428 1.00 60.43 N ATOM 21 CA GLU A 9 −5.171 −39.468 21.426 1.00 58.00 C ATOM 22 C GLU A 9 −6.208 −40.046 22.396 1.00 56.70 C ATOM 23 O GLU A 9 −7.195 −39.377 22.702 1.00 55.78 O ATOM 24 CB GLU A 9 −4.087 −38.688 22.193 1.00 60.92 C ATOM 25 CG GLU A 9 −2.938 −39.562 22.668 1.00 75.31 C ATOM 26 CD GLU A 9 −1.948 −39.953 21.584 1.00 104.77 C ATOM 27 OE1 GLU A 9 −1.472 −39.051 20.857 1.00 103.10 O ATOM 28 OE2 GLU A 9 −1.631 −41.160 21.478 1.00 98.24 O ATOM 29 N ARG A 10 −6.008 −41.310 22.814 1.00 50.38 N ATOM 30 CA ARG A 10 −6.901 −42.064 23.701 1.00 47.14 C ATOM 31 C ARG A 10 −8.282 −42.335 23.064 1.00 47.30 C ATOM 32 O ARG A 10 −9.236 −42.621 23.782 1.00 44.44 O ATOM 33 CB ARG A 10 −7.017 −41.411 25.101 1.00 48.88 C ATOM 34 CG ARG A 10 −5.713 −41.472 25.872 1.00 61.89 C ATOM 35 CD ARG A 10 −5.748 −40.691 27.166 1.00 73.75 C ATOM 36 NE ARG A 10 −4.476 −40.821 27.885 1.00 85.72 N ATOM 37 CZ ARG A 10 −4.204 −40.268 29.064 1.00 98.18 C ATOM 38 NH1 ARG A 10 −5.119 −39.529 29.691 1.00 79.44 N ATOM 39 NH2 ARG A 10 −3.018 −40.453 29.633 1.00 85.59 N ATOM 40 N HIS A 11 −8.373 −42.304 21.715 1.00 45.23 N ATOM 41 CA HIS A 11 −9.597 −42.625 20.976 1.00 44.43 C ATOM 42 C HIS A 11 −9.295 −43.733 19.975 1.00 48.94 C ATOM 43 O HIS A 11 −8.140 −43.927 19.608 1.00 50.15 O ATOM 44 CB HIS A 11 −10.152 −41.421 20.206 1.00 46.11 C ATOM 45 CG HIS A 11 −10.785 −40.363 21.043 1.00 49.09 C ATOM 46 ND1 HIS A 11 −10.052 −39.624 21.948 1.00 50.70 N ATOM 47 CD2 HIS A 11 −12.040 −39.866 20.990 1.00 51.51 C ATOM 48 CE1 HIS A 11 −10.893 −38.754 22.472 1.00 51.05 C ATOM 49 NE2 HIS A 11 −12.102 −38.849 21.907 1.00 51.94 N ATOM 50 N TYR A 12 −10.336 −44.426 19.504 1.00 44.82 N ATOM 51 CA TYR A 12 −10.199 −45.497 18.520 1.00 45.01 C ATOM 52 C TYR A 12 −11.328 −45.442 17.496 1.00 52.06 C ATOM 53 O TYR A 12 −12.419 −44.954 17.799 1.00 50.50 O ATOM 54 CB TYR A 12 −10.161 −46.868 19.208 1.00 42.79 C ATOM 55 CG TYR A 12 −11.478 −47.277 19.826 1.00 41.00 C ATOM 56 CD1 TYR A 12 −11.886 −46.756 21.048 1.00 40.15 C ATOM 57 CD2 TYR A 12 −12.293 −48.223 19.217 1.00 42.89 C ATOM 58 CE1 TYR A 12 −13.087 −47.144 21.633 1.00 38.61 C ATOM 59 CE2 TYR A 12 −13.516 −48.589 19.772 1.00 42.98 C ATOM 60 CZ TYR A 12 −13.906 −48.054 20.989 1.00 45.55 C ATOM 61 OH TYR A 12 −15.099 −48.416 21.564 1.00 42.17 O ATOM 62 N TRP A 13 −11.058 −45.961 16.291 1.00 51.96 N ATOM 63 CA TRP A 13 −12.029 −46.011 15.206 1.00 55.00 C ATOM 64 C TRP A 13 −12.959 −47.196 15.465 1.00 59.39 C ATOM 65 O TRP A 13 −12.486 −48.326 15.551 1.00 59.17 O ATOM 66 CB TRP A 13 −11.296 −46.159 13.868 1.00 57.28 C ATOM 67 CG TRP A 13 −12.151 −46.037 12.647 1.00 61.73 C ATOM 68 CD1 TRP A 13 −12.304 −46.971 11.665 1.00 67.19 C ATOM 69 CD2 TRP A 13 −12.844 −44.867 12.200 1.00 63.42 C ATOM 70 NE1 TRP A 13 −13.063 −46.459 10.641 1.00 69.98 N ATOM 71 CE2 TRP A 13 −13.420 −45.173 10.948 1.00 70.88 C ATOM 72 CE3 TRP A 13 −13.052 −43.594 12.744 1.00 64.03 C ATOM 73 CZ2 TRP A 13 −14.203 −44.257 10.240 1.00 72.61 C ATOM 74 CZ3 TRP A 13 −13.830 −42.685 12.046 1.00 68.06 C ATOM 75 CH2 TRP A 13 −14.389 −43.015 10.804 1.00 71.93 C ATOM 76 N ALA A 14 −14.270 −46.935 15.619 1.00 56.55 N ATOM 77 CA ALA A 14 −15.274 −47.957 15.920 1.00 56.51 C ATOM 78 C ALA A 14 −16.317 −48.081 14.818 1.00 64.02 C ATOM 79 O ALA A 14 −16.631 −47.090 14.156 1.00 65.10 O ATOM 80 CB ALA A 14 −15.981 −47.596 17.216 1.00 55.00 C ATOM 81 N GLN A 15 −16.865 −49.305 14.630 1.00 62.17 N ATOM 82 CA GLN A 15 −17.957 −49.591 13.685 1.00 65.61 C ATOM 83 C GLN A 15 −17.720 −49.083 12.241 1.00 73.48 C ATOM 84 O GLN A 15 −18.682 −49.002 11.474 1.00 76.16 O ATOM 85 CB GLN A 15 −19.279 −48.979 14.223 1.00 66.71 C ATOM 86 CG GLN A 15 −19.626 −49.315 15.687 1.00 78.93 C ATOM 87 CD GLN A 15 −20.419 −50.593 15.843 1.00 100.32 C ATOM 88 OE1 GLN A 15 −21.419 −50.817 15.153 1.00 97.14 O ATOM 89 NE2 GLN A 15 −20.050 −51.429 16.809 1.00 93.09 N ATOM 90 N GLY A 16 −16.462 −48.773 11.855 1.00 70.23 N ATOM 91 CA GLY A 16 −16.157 −48.222 10.534 1.00 73.11 C ATOM 92 C GLY A 16 −16.801 −46.837 10.313 1.00 77.68 C ATOM 93 O GLY A 16 −17.189 −46.543 9.185 1.00 80.88 O ATOM 94 N LYS A 17 −16.930 −45.997 11.376 1.00 71.01 N ATOM 95 CA LYS A 17 −17.599 −44.684 11.248 1.00 71.20 C ATOM 96 C LYS A 17 −17.369 −43.660 12.400 1.00 70.85 C ATOM 97 O LYS A 17 −17.370 −42.463 12.109 1.00 71.77 O ATOM 98 CB LYS A 17 −19.126 −44.858 11.030 1.00 74.62 C ATOM 99 CG LYS A 17 −19.751 −46.059 11.754 1.00 83.72 C ATOM 100 CD LYS A 17 −21.061 −45.782 12.501 1.00 89.88 C ATOM 101 CE LYS A 17 −22.179 −46.715 12.096 1.00 99.90 C ATOM 102 NZ LYS A 17 −23.332 −46.635 13.031 1.00 105.06 N ATOM 103 N LEU A 18 −17.239 −44.092 13.673 1.00 63.17 N ATOM 104 CA LEU A 18 −17.067 −43.180 14.817 1.00 60.28 C ATOM 105 C LEU A 18 −15.676 −43.242 15.444 1.00 61.62 C ATOM 106 O LEU A 18 −15.061 −44.302 15.437 1.00 60.54 O ATOM 107 CB LEU A 18 −18.060 −43.566 15.930 1.00 58.45 C ATOM 108 CG LEU A 18 −19.518 −43.752 15.549 1.00 64.20 C ATOM 109 CD1 LEU A 18 −20.310 −44.282 16.718 1.00 62.59 C ATOM 110 CD2 LEU A 18 −20.120 −42.474 15.073 1.00 67.55 C ATOM 111 N CYS A 19 −15.231 −42.134 16.073 1.00 57.40 N ATOM 112 CA CYS A 19 −14.017 −42.113 16.893 1.00 56.18 C ATOM 113 C CYS A 19 −14.558 −42.153 18.324 1.00 54.32 C ATOM 114 O CYS A 19 −15.261 −41.229 18.706 1.00 53.32 O ATOM 115 CB CYS A 19 −13.153 −40.875 16.660 1.00 59.52 C ATOM 116 SG CYS A 19 −12.060 −40.981 15.212 1.00 67.70 S ATOM 117 N CYS A 20 −14.304 −43.237 19.079 1.00 47.18 N ATOM 118 CA CYS A 20 −14.785 −43.396 20.461 1.00 43.78 C ATOM 119 C CYS A 20 −13.637 −43.186 21.402 1.00 43.86 C ATOM 120 O CYS A 20 −12.522 −43.552 21.050 1.00 41.32 O ATOM 121 CB CYS A 20 −15.364 −44.794 20.661 1.00 43.12 C ATOM 122 SG CYS A 20 −16.797 −45.180 19.633 1.00 48.76 S ATOM 123 N GLN A 21 −13.903 −42.707 22.636 1.00 40.51 N ATOM 124 CA GLN A 21 −12.846 −42.621 23.647 1.00 39.59 C ATOM 125 C GLN A 21 −12.604 −44.033 24.119 1.00 40.37 C ATOM 126 O GLN A 21 −13.565 −44.777 24.325 1.00 39.21 O ATOM 127 CB GLN A 21 −13.240 −41.788 24.880 1.00 41.38 C ATOM 128 CG GLN A 21 −12.957 −40.314 24.748 1.00 64.78 C ATOM 129 CD GLN A 21 −14.126 −39.437 25.123 1.00 83.23 C ATOM 130 OE1 GLN A 21 −14.726 −39.596 26.194 1.00 80.48 O ATOM 131 NE2 GLN A 21 −14.459 −38.470 24.265 1.00 73.73 N ATOM 132 N MET A 22 −11.338 −44.391 24.336 1.00 36.78 N ATOM 133 CA MET A 22 −10.980 −45.711 24.845 1.00 36.42 C ATOM 134 C MET A 22 −11.432 −45.844 26.301 1.00 39.59 C ATOM 135 O MET A 22 −11.712 −44.845 26.972 1.00 38.93 O ATOM 136 CB MET A 22 −9.454 −45.921 24.795 1.00 38.78 C ATOM 137 CG MET A 22 −8.878 −45.994 23.402 1.00 44.78 C ATOM 138 SD MET A 22 −7.091 −46.261 23.563 1.00 50.58 S ATOM 139 CE MET A 22 −6.482 −45.469 22.164 1.00 49.85 C ATOM 140 N CYS A 23 −11.460 −47.073 26.792 1.00 36.74 N ATOM 141 CA CYS A 23 −11.785 −47.338 28.191 1.00 35.73 C ATOM 142 C CYS A 23 −10.649 −46.865 29.056 1.00 36.92 C ATOM 143 O CYS A 23 −9.498 −47.012 28.673 1.00 35.59 O ATOM 144 CB CYS A 23 −12.044 −48.822 28.415 1.00 36.05 C ATOM 145 SG CYS A 23 −13.586 −49.417 27.700 1.00 41.15 S ATOM 146 N GLU A 24 −10.964 −46.331 30.229 1.00 34.16 N ATOM 147 CA GLU A 24 −9.950 −45.886 31.179 1.00 34.33 C ATOM 148 C GLU A 24 −9.256 −47.092 31.827 1.00 35.25 C ATOM 149 O GLU A 24 −9.836 −48.180 31.837 1.00 32.32 O ATOM 150 CB GLU A 24 −10.605 −45.049 32.295 1.00 36.84 C ATOM 151 CG GLU A 24 −11.093 −43.686 31.819 1.00 52.17 C ATOM 152 CD GLU A 24 −10.002 −42.654 31.608 1.00 82.21 C ATOM 153 OE1 GLU A 24 −9.189 −42.452 32.539 1.00 87.94 O ATOM 154 OE2 GLU A 24 −9.970 −42.030 30.521 1.00 83.01 O ATOM 155 N PRO A 25 −8.053 −46.898 32.420 1.00 33.02 N ATOM 156 CA PRO A 25 −7.392 −47.965 33.173 1.00 32.41 C ATOM 157 C PRO A 25 −8.331 −48.393 34.301 1.00 34.90 C ATOM 158 O PRO A 25 −9.005 −47.546 34.873 1.00 33.20 O ATOM 159 CB PRO A 25 −6.141 −47.284 33.739 1.00 34.85 C ATOM 160 CG PRO A 25 −5.887 −46.136 32.862 1.00 38.86 C ATOM 161 CD PRO A 25 −7.170 −45.723 32.250 1.00 35.27 C ATOM 162 N GLY A 26 −8.426 −49.690 34.577 1.00 32.28 N ATOM 163 CA GLY A 26 −9.314 −50.180 35.629 1.00 32.48 C ATOM 164 C GLY A 26 −10.737 −50.440 35.161 1.00 34.53 C ATOM 165 O GLY A 26 −11.589 −50.726 35.992 1.00 34.04 O ATOM 166 N THR A 27 −10.993 −50.381 33.846 1.00 30.91 N ATOM 167 CA THR A 27 −12.302 −50.696 33.268 1.00 31.44 C ATOM 168 C THR A 27 −12.086 −51.594 32.038 1.00 35.38 C ATOM 169 O THR A 27 −10.942 −51.852 31.650 1.00 35.01 O ATOM 170 CB THR A 27 −13.102 −49.401 32.910 1.00 33.54 C ATOM 171 OG1 THR A 27 −12.552 −48.797 31.743 1.00 33.25 O ATOM 172 CG2 THR A 27 −13.142 −48.377 34.046 1.00 26.78 C ATOM 173 N PHE A 28 −13.178 −52.084 31.443 1.00 31.97 N ATOM 174 CA PHE A 28 −13.122 −52.897 30.227 1.00 31.35 C ATOM 175 C PHE A 28 −14.306 −52.547 29.352 1.00 36.32 C ATOM 176 O PHE A 28 −15.330 −52.105 29.859 1.00 36.35 O ATOM 177 CB PHE A 28 −13.080 −54.403 30.520 1.00 32.57 C ATOM 178 CG PHE A 28 −14.347 −54.983 31.102 1.00 34.95 C ATOM 179 CD1 PHE A 28 −14.618 −54.885 32.464 1.00 37.23 C ATOM 180 CD2 PHE A 28 −15.246 −55.675 30.301 1.00 37.68 C ATOM 181 CE1 PHE A 28 −15.787 −55.432 33.002 1.00 38.93 C ATOM 182 CE2 PHE A 28 −16.416 −56.219 30.841 1.00 41.16 C ATOM 183 CZ PHE A 28 −16.673 −56.101 32.188 1.00 38.96 C ATOM 184 N LEU A 29 −14.177 −52.792 28.056 1.00 35.00 N ATOM 185 CA LEU A 29 −15.199 −52.441 27.074 1.00 36.44 C ATOM 186 C LEU A 29 −16.403 −53.371 27.033 1.00 42.33 C ATOM 187 O LEU A 29 −16.258 −54.578 26.833 1.00 43.83 O ATOM 188 CB LEU A 29 −14.563 −52.362 25.676 1.00 37.20 C ATOM 189 CG LEU A 29 −15.406 −51.618 24.627 1.00 42.73 C ATOM 190 CD1 LEU A 29 −14.775 −50.323 24.246 1.00 42.68 C ATOM 191 CD2 LEU A 29 −15.627 −52.456 23.414 1.00 44.91 C ATOM 192 N VAL A 30 −17.604 −52.793 27.202 1.00 39.41 N ATOM 193 CA VAL A 30 −18.871 −53.513 27.085 1.00 41.21 C ATOM 194 C VAL A 30 −19.482 −53.213 25.707 1.00 46.02 C ATOM 195 O VAL A 30 −19.950 −54.128 25.034 1.00 47.76 O ATOM 196 CB VAL A 30 −19.837 −53.135 28.228 1.00 45.82 C ATOM 197 CG1 VAL A 30 −21.221 −53.759 28.016 1.00 47.98 C ATOM 198 CG2 VAL A 30 −19.263 −53.567 29.566 1.00 45.02 C ATOM 199 N LYS A 31 −19.509 −51.938 25.316 1.00 41.83 N ATOM 200 CA LYS A 31 −20.091 −51.500 24.052 1.00 43.31 C ATOM 201 C LYS A 31 −19.307 −50.299 23.527 1.00 43.94 C ATOM 202 O LYS A 31 −18.775 −49.519 24.321 1.00 41.40 O ATOM 203 CB LYS A 31 −21.564 −51.086 24.305 1.00 48.54 C ATOM 204 CG LYS A 31 −22.522 −51.318 23.139 1.00 71.10 C ATOM 205 CD LYS A 31 −23.697 −52.236 23.506 1.00 87.51 C ATOM 206 CE LYS A 31 −23.296 −53.686 23.667 1.00 96.81 C ATOM 207 NZ LYS A 31 −24.467 −54.549 23.980 1.00 106.62 N ATOM 208 N ASP A 32 −19.256 −50.127 22.202 1.00 40.83 N ATOM 209 CA ASP A 32 −18.613 −48.948 21.603 1.00 40.22 C ATOM 210 C ASP A 32 −19.469 −47.713 21.890 1.00 45.15 C ATOM 211 O ASP A 32 −20.641 −47.833 22.274 1.00 44.59 O ATOM 212 CB ASP A 32 −18.503 −49.092 20.069 1.00 43.48 C ATOM 213 CG ASP A 32 −17.539 −50.151 19.586 1.00 50.06 C ATOM 214 OD1 ASP A 32 −16.467 −50.308 20.212 1.00 48.33 O ATOM 215 OD2 ASP A 32 −17.808 −50.757 18.531 1.00 54.24 O ATOM 216 N CYS A 33 −18.911 −46.523 21.639 1.00 43.00 N ATOM 217 CA CYS A 33 −19.680 −45.287 21.764 1.00 43.55 C ATOM 218 C CYS A 33 −20.714 −45.314 20.622 1.00 47.10 C ATOM 219 O CYS A 33 −20.476 −45.974 19.607 1.00 47.00 O ATOM 220 CB CYS A 33 −18.770 −44.056 21.690 1.00 43.96 C ATOM 221 SG CYS A 33 −18.121 −43.687 20.036 1.00 49.80 S ATOM 222 N ASP A 34 −21.875 −44.677 20.824 1.00 43.01 N ATOM 223 CA ASP A 34 −22.940 −44.589 19.811 1.00 44.20 C ATOM 224 C ASP A 34 −22.996 −43.161 19.209 1.00 48.29 C ATOM 225 O ASP A 34 −23.871 −42.862 18.387 1.00 48.66 O ATOM 226 CB ASP A 34 −24.308 −45.011 20.406 1.00 46.75 C ATOM 227 CG ASP A 34 −24.754 −44.274 21.661 1.00 57.67 C ATOM 228 OD1 ASP A 34 −24.019 −43.373 22.118 1.00 58.91 O ATOM 229 OD2 ASP A 34 −25.801 −44.641 22.221 1.00 60.93 O ATOM 230 N GLN A 35 −22.065 −42.278 19.638 1.00 42.81 N ATOM 231 CA GLN A 35 −21.969 −40.893 19.188 1.00 43.04 C ATOM 232 C GLN A 35 −20.485 −40.598 18.935 1.00 45.91 C ATOM 233 O GLN A 35 −19.647 −40.986 19.741 1.00 43.23 O ATOM 234 CB GLN A 35 −22.510 −39.951 20.277 1.00 43.90 C ATOM 235 CG GLN A 35 −23.986 −40.127 20.619 1.00 41.63 C ATOM 236 CD GLN A 35 −24.919 −39.587 19.576 1.00 51.47 C ATOM 237 OE1 GLN A 35 −25.450 −38.489 19.713 1.00 49.14 O ATOM 238 NE2 GLN A 35 −25.221 −40.364 18.548 1.00 46.18 N ATOM 239 N HIS A 36 −20.166 −39.904 17.842 1.00 45.31 N ATOM 240 CA HIS A 36 −18.788 −39.563 17.482 1.00 44.53 C ATOM 241 C HIS A 36 −18.101 −38.713 18.574 1.00 47.23 C ATOM 242 O HIS A 36 −18.690 −37.769 19.090 1.00 45.88 O ATOM 243 CB HIS A 36 −18.790 −38.846 16.126 1.00 47.91 C ATOM 244 CG HIS A 36 −17.451 −38.404 15.633 1.00 51.77 C ATOM 245 ND1 HIS A 36 −16.450 −39.309 15.324 1.00 52.55 N ATOM 246 CD2 HIS A 36 −17.005 −37.156 15.386 1.00 54.50 C ATOM 247 CE1 HIS A 36 −15.436 −38.581 14.881 1.00 53.18 C ATOM 248 NE2 HIS A 36 −15.732 −37.276 14.894 1.00 54.90 N ATOM 249 N ARG A 37 −16.864 −39.087 18.935 1.00 44.51 N ATOM 250 CA ARG A 37 −16.036 −38.444 19.963 1.00 43.70 C ATOM 251 C ARG A 37 −16.686 −38.429 21.363 1.00 47.06 C ATOM 252 O ARG A 37 −16.444 −37.518 22.160 1.00 47.70 O ATOM 253 CB ARG A 37 −15.581 −37.047 19.522 1.00 45.20 C ATOM 254 CG ARG A 37 −14.521 −37.113 18.440 1.00 48.90 C ATOM 255 CD ARG A 37 −13.864 −35.772 18.225 1.00 49.68 C ATOM 256 NE ARG A 37 −12.902 −35.824 17.124 1.00 50.76 N ATOM 257 CZ ARG A 37 −11.978 −34.901 16.869 1.00 65.51 C ATOM 258 NH1 ARG A 37 −11.881 −33.816 17.634 1.00 55.67 N ATOM 259 NH2 ARG A 37 −11.146 −35.053 15.848 1.00 60.89 N ATOM 260 N LYS A 38 −17.442 −39.486 21.684 1.00 42.28 N ATOM 261 CA LYS A 38 −18.019 −39.683 23.013 1.00 41.17 C ATOM 262 C LYS A 38 −17.441 −40.974 23.596 1.00 43.23 C ATOM 263 O LYS A 38 −16.715 −41.701 22.911 1.00 42.89 O ATOM 264 CB LYS A 38 −19.555 −39.684 22.981 1.00 43.69 C ATOM 265 CG LYS A 38 −20.156 −38.361 22.520 1.00 43.89 C ATOM 266 CD LYS A 38 −19.885 −37.206 23.482 1.00 42.15 C ATOM 267 CE LYS A 38 −20.455 −35.924 22.978 1.00 36.14 C ATOM 268 NZ LYS A 38 −19.792 −34.752 23.603 1.00 41.15 N ATOM 269 N ALA A 39 −17.700 −41.223 24.871 1.00 39.17 N ATOM 270 CA ALA A 39 −17.129 −42.380 25.560 1.00 37.31 C ATOM 271 C ALA A 39 −17.765 −43.711 25.213 1.00 41.29 C ATOM 272 O ALA A 39 −18.984 −43.808 25.053 1.00 42.27 O ATOM 273 CB ALA A 39 −17.192 −42.166 27.067 1.00 37.51 C ATOM 274 N ALA A 40 −16.926 −44.756 25.145 1.00 37.60 N ATOM 275 CA ALA A 40 −17.382 −46.141 24.997 1.00 36.76 C ATOM 276 C ALA A 40 −18.008 −46.529 26.340 1.00 39.81 C ATOM 277 O ALA A 40 −17.688 −45.906 27.355 1.00 37.62 O ATOM 278 CB ALA A 40 −16.195 −47.061 24.707 1.00 35.94 C ATOM 279 N GLN A 41 −18.897 −47.535 26.359 1.00 37.98 N ATOM 280 CA GLN A 41 −19.507 −47.988 27.610 1.00 37.92 C ATOM 281 C GLN A 41 −18.517 −48.991 28.230 1.00 39.59 C ATOM 282 O GLN A 41 −18.250 −50.031 27.638 1.00 36.25 O ATOM 283 CB GLN A 41 −20.886 −48.626 27.360 1.00 41.38 C ATOM 284 CG GLN A 41 −21.717 −48.786 28.636 1.00 59.82 C ATOM 285 CD GLN A 41 −22.262 −50.180 28.824 1.00 80.54 C ATOM 286 OE1 GLN A 41 −21.904 −50.886 29.773 1.00 78.64 O ATOM 287 NE2 GLN A 41 −23.147 −50.605 27.931 1.00 71.47 N ATOM 288 N CYS A 42 −17.903 −48.622 29.363 1.00 37.80 N ATOM 289 CA CYS A 42 −16.889 −49.436 30.005 1.00 37.75 C ATOM 290 C CYS A 42 −17.290 −49.772 31.424 1.00 41.34 C ATOM 291 O CYS A 42 −17.585 −48.858 32.192 1.00 41.87 O ATOM 292 CB CYS A 42 −15.541 −48.716 29.983 1.00 37.77 C ATOM 293 SG CYS A 42 −15.030 −48.137 28.345 1.00 42.12 S ATOM 294 N ASP A 43 −17.211 −51.059 31.800 1.00 37.48 N ATOM 295 CA ASP A 43 −17.531 −51.521 33.159 1.00 37.21 C ATOM 296 C ASP A 43 −16.249 −51.620 33.983 1.00 37.85 C ATOM 297 O ASP A 43 −15.192 −51.862 33.412 1.00 35.75 O ATOM 298 CB ASP A 43 −18.213 −52.907 33.130 1.00 40.32 C ATOM 299 CG ASP A 43 −19.722 −52.906 32.975 1.00 55.05 C ATOM 300 OD1 ASP A 43 −20.321 −51.809 32.955 1.00 59.52 O ATOM 301 OD2 ASP A 43 −20.303 −54.002 32.841 1.00 59.29 O ATOM 302 N PRO A 44 −16.333 −51.504 35.321 1.00 35.04 N ATOM 303 CA PRO A 44 −15.117 −51.616 36.138 1.00 33.83 C ATOM 304 C PRO A 44 −14.507 −53.016 36.211 1.00 37.49 C ATOM 305 O PRO A 44 −15.222 −54.021 36.168 1.00 37.52 O ATOM 306 CB PRO A 44 −15.595 −51.212 37.536 1.00 36.41 C ATOM 307 CG PRO A 44 −17.038 −51.530 37.556 1.00 41.52 C ATOM 308 CD PRO A 44 −17.529 −51.281 36.164 1.00 36.85 C ATOM 309 N CYS A 45 −13.196 −53.069 36.466 1.00 34.81 N ATOM 310 CA CYS A 45 −12.497 −54.322 36.755 1.00 35.43 C ATOM 311 C CYS A 45 −12.849 −54.662 38.213 1.00 37.20 C ATOM 312 O CYS A 45 −13.441 −53.836 38.916 1.00 36.50 O ATOM 313 CB CYS A 45 −10.986 −54.181 36.570 1.00 35.93 C ATOM 314 SG CYS A 45 −10.456 −53.899 34.863 1.00 39.57 S ATOM 315 N ILE A 46 −12.483 −55.852 38.673 1.00 33.32 N ATOM 316 CA ILE A 46 −12.824 −56.299 40.028 1.00 34.48 C ATOM 317 C ILE A 46 −11.638 −56.144 40.981 1.00 38.11 C ATOM 318 O ILE A 46 −10.671 −56.889 40.827 1.00 37.43 O ATOM 319 CB ILE A 46 −13.317 −57.764 39.960 1.00 38.33 C ATOM 320 CG1 ILE A 46 −14.418 −57.947 38.887 1.00 38.73 C ATOM 321 CG2 ILE A 46 −13.786 −58.241 41.335 1.00 39.59 C ATOM 322 CD1 ILE A 46 −15.654 −57.116 39.054 1.00 47.23 C ATOM 323 N PRO A 47 −11.712 −55.225 41.992 1.00 36.72 N ATOM 324 CA PRO A 47 −10.594 −55.057 42.940 1.00 37.81 C ATOM 325 C PRO A 47 −10.144 −56.388 43.545 1.00 41.77 C ATOM 326 O PRO A 47 −10.973 −57.120 44.084 1.00 42.22 O ATOM 327 CB PRO A 47 −11.154 −54.108 44.017 1.00 40.91 C ATOM 328 CG PRO A 47 −12.248 −53.380 43.361 1.00 44.71 C ATOM 329 CD PRO A 47 −12.804 −54.272 42.279 1.00 38.78 C ATOM 330 N GLY A 48 −8.854 −56.735 43.375 1.00 38.47 N ATOM 331 CA GLY A 48 −8.281 −57.975 43.910 1.00 39.27 C ATOM 332 C GLY A 48 −8.430 −59.204 42.993 1.00 42.73 C ATOM 333 O GLY A 48 −7.886 −60.242 43.332 1.00 43.38 O ATOM 334 N VAL A 49 −9.156 −59.100 41.854 1.00 40.03 N ATOM 335 CA VAL A 49 −9.382 −60.230 40.925 1.00 40.03 C ATOM 336 C VAL A 49 −8.969 −59.918 39.489 1.00 40.57 C ATOM 337 O VAL A 49 −8.412 −60.789 38.830 1.00 39.24 O ATOM 338 CB VAL A 49 −10.860 −60.693 41.005 1.00 45.05 C ATOM 339 CG1 VAL A 49 −11.158 −61.823 40.016 1.00 45.50 C ATOM 340 CG2 VAL A 49 −11.206 −61.101 42.427 1.00 46.81 C ATOM 341 N SER A 50 −9.234 −58.705 38.990 1.00 35.94 N ATOM 342 CA SER A 50 −8.838 −58.347 37.633 1.00 33.71 C ATOM 343 C SER A 50 −8.374 −56.902 37.528 1.00 35.99 C ATOM 344 O SER A 50 −8.600 −56.099 38.434 1.00 36.25 O ATOM 345 CB SER A 50 −9.968 −58.657 36.655 1.00 36.17 C ATOM 346 OG SER A 50 −11.095 −57.827 36.872 1.00 38.18 O ATOM 347 N PHE A 51 −7.659 −56.583 36.445 1.00 32.04 N ATOM 348 CA PHE A 51 −7.095 −55.250 36.261 1.00 29.14 C ATOM 349 C PHE A 51 −6.901 −54.890 34.799 1.00 32.56 C ATOM 350 O PHE A 51 −6.971 −55.746 33.935 1.00 31.66 O ATOM 351 CB PHE A 51 −5.739 −55.175 36.993 1.00 29.13 C ATOM 352 CG PHE A 51 −4.630 −55.964 36.343 1.00 28.17 C ATOM 353 CD1 PHE A 51 −4.511 −57.333 36.554 1.00 29.81 C ATOM 354 CD2 PHE A 51 −3.696 −55.338 35.529 1.00 27.50 C ATOM 355 CE1 PHE A 51 −3.504 −58.066 35.922 1.00 29.84 C ATOM 356 CE2 PHE A 51 −2.716 −56.079 34.867 1.00 28.98 C ATOM 357 CZ PHE A 51 −2.619 −57.435 35.078 1.00 27.36 C ATOM 358 N SER A 52 −6.631 −53.620 34.533 1.00 29.38 N ATOM 359 CA SER A 52 −6.296 −53.147 33.189 1.00 29.06 C ATOM 360 C SER A 52 −5.409 −51.902 33.393 1.00 32.84 C ATOM 361 O SER A 52 −5.882 −50.907 33.934 1.00 32.41 O ATOM 362 CB SER A 52 −7.539 −52.889 32.342 1.00 30.73 C ATOM 363 OG SER A 52 −8.389 −51.920 32.922 1.00 37.68 O ATOM 364 N PRO A 53 −4.098 −52.008 33.095 1.00 29.99 N ATOM 365 CA PRO A 53 −3.162 −50.916 33.381 1.00 30.41 C ATOM 366 C PRO A 53 −3.249 −49.652 32.542 1.00 35.49 C ATOM 367 O PRO A 53 −2.704 −48.641 32.978 1.00 35.51 O ATOM 368 CB PRO A 53 −1.783 −51.567 33.197 1.00 32.77 C ATOM 369 CG PRO A 53 −2.002 −52.773 32.375 1.00 35.90 C ATOM 370 CD PRO A 53 −3.438 −53.136 32.400 1.00 30.40 C ATOM 371 N ASP A 54 −3.868 −49.678 31.363 1.00 34.35 N ATOM 372 CA ASP A 54 −3.878 −48.483 30.524 1.00 36.60 C ATOM 373 C ASP A 54 −5.175 −48.300 29.750 1.00 38.50 C ATOM 374 O ASP A 54 −6.066 −49.142 29.842 1.00 38.49 O ATOM 375 CB ASP A 54 −2.668 −48.523 29.570 1.00 41.23 C ATOM 376 CG ASP A 54 −1.990 −47.160 29.419 1.00 62.91 C ATOM 377 OD1 ASP A 54 −2.649 −46.223 28.921 1.00 63.05 O ATOM 378 OD2 ASP A 54 −0.827 −47.016 29.872 1.00 76.03 O ATOM 379 N HIS A 55 −5.282 −47.179 29.010 1.00 33.79 N ATOM 380 CA HIS A 55 −6.426 −46.889 28.145 1.00 33.82 C ATOM 381 C HIS A 55 −6.454 −47.966 27.055 1.00 36.99 C ATOM 382 O HIS A 55 −5.406 −48.281 26.495 1.00 37.92 O ATOM 383 CB HIS A 55 −6.320 −45.480 27.517 1.00 35.97 C ATOM 384 CG HIS A 55 −6.435 −44.397 28.536 1.00 39.79 C ATOM 385 ND1 HIS A 55 −5.336 −43.965 29.260 1.00 42.24 N ATOM 386 CD2 HIS A 55 −7.530 −43.735 28.969 1.00 41.07 C ATOM 387 CE1 HIS A 55 −5.795 −43.065 30.110 1.00 42.04 C ATOM 388 NE2 HIS A 55 −7.108 −42.880 29.956 1.00 42.15 N ATOM 389 N HIS A 56 −7.606 −48.587 26.817 1.00 32.75 N ATOM 390 CA HIS A 56 −7.665 −49.715 25.896 1.00 33.13 C ATOM 391 C HIS A 56 −9.080 −49.974 25.395 1.00 37.42 C ATOM 392 O HIS A 56 −10.019 −49.303 25.805 1.00 37.11 O ATOM 393 CB HIS A 56 −7.122 −50.983 26.615 1.00 32.46 C ATOM 394 CG HIS A 56 −8.064 −51.522 27.648 1.00 33.84 C ATOM 395 ND1 HIS A 56 −8.814 −52.647 27.409 1.00 34.70 N ATOM 396 CD2 HIS A 56 −8.447 −50.985 28.830 1.00 34.33 C ATOM 397 CE1 HIS A 56 −9.559 −52.816 28.487 1.00 33.79 C ATOM 398 NE2 HIS A 56 −9.361 −51.844 29.372 1.00 33.76 N ATOM 399 N THR A 57 −9.211 −50.968 24.520 1.00 35.36 N ATOM 400 CA THR A 57 −10.474 −51.373 23.912 1.00 36.44 C ATOM 401 C THR A 57 −10.748 −52.884 24.091 1.00 40.44 C ATOM 402 O THR A 57 −11.658 −53.395 23.436 1.00 42.37 O ATOM 403 CB THR A 57 −10.449 −50.990 22.418 1.00 39.91 C ATOM 404 OG1 THR A 57 −9.388 −51.692 21.770 1.00 42.78 O ATOM 405 CG2 THR A 57 −10.273 −49.506 22.204 1.00 36.11 C ATOM 406 N ARG A 58 −10.019 −53.597 24.984 1.00 36.40 N ATOM 407 CA ARG A 58 −10.256 −55.047 25.161 1.00 36.92 C ATOM 408 C ARG A 58 −11.596 −55.265 25.871 1.00 40.95 C ATOM 409 O ARG A 58 −11.891 −54.528 26.812 1.00 40.53 O ATOM 410 CB ARG A 58 −9.116 −55.740 25.960 1.00 34.53 C ATOM 411 CG ARG A 58 −7.699 −55.544 25.419 1.00 35.34 C ATOM 412 CD ARG A 58 −7.592 −55.879 23.949 1.00 43.83 C ATOM 413 NE ARG A 58 −7.909 −57.298 23.720 1.00 49.81 N ATOM 414 CZ ARG A 58 −8.709 −57.793 22.775 1.00 59.77 C ATOM 415 NH1 ARG A 58 −9.312 −56.980 21.905 1.00 47.98 N ATOM 416 NH2 ARG A 58 −8.909 −59.102 22.686 1.00 50.31 N ATOM 417 N PRO A 59 −12.387 −56.280 25.471 1.00 38.74 N ATOM 418 CA PRO A 59 −13.675 −56.527 26.131 1.00 38.89 C ATOM 419 C PRO A 59 −13.581 −57.357 27.434 1.00 41.04 C ATOM 420 O PRO A 59 −14.534 −58.047 27.805 1.00 41.72 O ATOM 421 CB PRO A 59 −14.495 −57.223 25.036 1.00 42.78 C ATOM 422 CG PRO A 59 −13.492 −57.974 24.269 1.00 48.48 C ATOM 423 CD PRO A 59 −12.275 −57.075 24.230 1.00 42.93 C ATOM 424 N HIS A 60 −12.463 −57.243 28.155 1.00 35.45 N ATOM 425 CA HIS A 60 −12.248 −57.925 29.423 1.00 34.60 C ATOM 426 C HIS A 60 −11.118 −57.219 30.162 1.00 38.20 C ATOM 427 O HIS A 60 −10.394 −56.403 29.580 1.00 36.57 O ATOM 428 CB HIS A 60 −11.878 −59.419 29.190 1.00 36.05 C ATOM 429 CG HIS A 60 −10.628 −59.598 28.379 1.00 38.86 C ATOM 430 ND1 HIS A 60 −10.683 −59.824 27.021 1.00 41.96 N ATOM 431 CD2 HIS A 60 −9.330 −59.468 28.741 1.00 38.40 C ATOM 432 CE1 HIS A 60 −9.427 −59.872 26.608 1.00 40.51 C ATOM 433 NE2 HIS A 60 −8.578 −59.662 27.614 1.00 38.97 N ATOM 434 N CYS A 61 −10.957 −57.573 31.432 1.00 37.29 N ATOM 435 CA CYS A 61 −9.859 −57.125 32.279 1.00 36.48 C ATOM 436 C CYS A 61 −8.856 −58.274 32.319 1.00 37.87 C ATOM 437 O CYS A 61 −9.229 −59.415 32.092 1.00 37.13 O ATOM 438 CB CYS A 61 −10.354 −56.790 33.684 1.00 37.27 C ATOM 439 SG CYS A 61 −11.404 −55.331 33.760 1.00 41.49 S ATOM 440 N GLU A 62 −7.606 −57.977 32.650 1.00 34.49 N ATOM 441 CA GLU A 62 −6.542 −58.977 32.805 1.00 33.63 C ATOM 442 C GLU A 62 −6.763 −59.657 34.142 1.00 36.33 C ATOM 443 O GLU A 62 −7.114 −58.982 35.102 1.00 34.77 O ATOM 444 CB GLU A 62 −5.164 −58.291 32.836 1.00 33.97 C ATOM 445 CG GLU A 62 −4.864 −57.415 31.631 1.00 36.09 C ATOM 446 CD GLU A 62 −4.928 −58.187 30.334 1.00 43.71 C ATOM 447 OE1 GLU A 62 −6.009 −58.203 29.706 1.00 37.36 O ATOM 448 OE2 GLU A 62 −3.937 −58.878 30.015 1.00 34.40 O ATOM 449 N SER A 63 −6.557 −60.975 34.227 1.00 33.89 N ATOM 450 CA SER A 63 −6.722 −61.674 35.493 1.00 33.61 C ATOM 451 C SER A 63 −5.503 −61.426 36.363 1.00 37.78 C ATOM 452 O SER A 63 −4.378 −61.525 35.871 1.00 37.22 O ATOM 453 CB SER A 63 −6.867 −63.178 35.265 1.00 37.53 C ATOM 454 OG SER A 63 −8.072 −63.491 34.590 1.00 44.08 O ATOM 455 N CYS A 64 −5.715 −61.139 37.654 1.00 35.84 N ATOM 456 CA CYS A 64 −4.615 −61.014 38.609 1.00 38.42 C ATOM 457 C CYS A 64 −3.997 −62.394 38.798 1.00 43.03 C ATOM 458 O CYS A 64 −4.741 −63.369 38.902 1.00 43.07 O ATOM 459 CB CYS A 64 −5.112 −60.488 39.953 1.00 41.22 C ATOM 460 SG CYS A 64 −5.968 −58.900 39.863 1.00 45.41 S ATOM 461 N ARG A 65 −2.659 −62.474 38.917 1.00 40.94 N ATOM 462 CA ARG A 65 −1.981 −63.739 39.226 1.00 42.73 C ATOM 463 C ARG A 65 −2.246 −64.060 40.699 1.00 50.59 C ATOM 464 O ARG A 65 −2.498 −63.143 41.488 1.00 47.96 O ATOM 465 CB ARG A 65 −0.462 −63.660 38.966 1.00 42.53 C ATOM 466 CG ARG A 65 0.328 −62.666 39.849 1.00 44.59 C ATOM 467 CD ARG A 65 1.821 −62.847 39.684 1.00 47.03 C ATOM 468 NE ARG A 65 2.234 −62.737 38.282 1.00 41.68 N ATOM 469 CZ ARG A 65 2.512 −61.615 37.620 1.00 45.98 C ATOM 470 NH1 ARG A 65 2.432 −60.431 38.231 1.00 35.56 N ATOM 471 NH2 ARG A 65 2.872 −61.664 36.345 1.00 34.94 N ATOM 472 N HIS A 66 −2.181 −65.350 41.064 1.00 53.75 N ATOM 473 CA HIS A 66 −2.415 −65.809 42.436 1.00 57.95 C ATOM 474 C HIS A 66 −1.089 −66.172 43.088 1.00 60.73 C ATOM 475 O HIS A 66 −0.259 −66.829 42.457 1.00 60.16 O ATOM 476 CB HIS A 66 −3.344 −67.033 42.446 1.00 62.92 C ATOM 477 CG HIS A 66 −4.512 −66.902 41.518 1.00 67.42 C ATOM 478 ND1 HIS A 66 −4.607 −67.669 40.365 1.00 70.55 N ATOM 479 CD2 HIS A 66 −5.569 −66.057 41.571 1.00 69.55 C ATOM 480 CE1 HIS A 66 −5.726 −67.288 39.768 1.00 69.97 C ATOM 481 NE2 HIS A 66 −6.340 −66.318 40.459 1.00 69.56 N ATOM 482 N CYS A 67 −0.895 −65.750 44.350 1.00 57.25 N ATOM 483 CA CYS A 67 0.307 −66.047 45.120 1.00 58.83 C ATOM 484 C CYS A 67 −0.065 −67.179 46.096 1.00 71.24 C ATOM 485 O CYS A 67 −0.155 −66.958 47.301 1.00 72.76 O ATOM 486 CB CYS A 67 0.800 −64.797 45.851 1.00 57.41 C ATOM 487 SG CYS A 67 1.035 −63.346 44.781 1.00 57.53 S ATOM 488 N ASN A 68 −0.340 −68.383 45.546 1.00 72.87 N ATOM 489 CA ASN A 68 −0.744 −69.550 46.358 1.00 78.32 C ATOM 490 C ASN A 68 0.421 −70.074 47.203 1.00 87.39 C ATOM 491 O ASN A 68 1.429 −70.522 46.652 1.00 87.06 O ATOM 492 CB ASN A 68 −1.361 −70.678 45.504 1.00 83.46 C ATOM 493 CG ASN A 68 −0.515 −71.155 44.343 1.00 111.51 C ATOM 494 OD1 ASN A 68 −0.601 −70.625 43.227 1.00 104.57 O ATOM 495 ND2 ASN A 68 0.353 −72.132 44.589 1.00 105.22 N ATOM 496 O SER A 69 2.613 −68.877 50.926 1.00 97.30 O ATOM 497 N SER A 69 0.300 −69.956 48.545 1.00 88.43 N ATOM 498 CA SER A 69 1.305 −70.337 49.557 1.00 92.23 C ATOM 499 C SER A 69 2.319 −69.208 49.775 1.00 94.76 C ATOM 500 CB SER A 69 1.998 −71.668 49.246 1.00 99.50 C ATOM 501 OG SER A 69 3.179 −71.523 48.471 1.00 108.86 O ATOM 502 N GLY A 70 2.846 −68.613 48.684 1.00 86.65 N ATOM 503 CA GLY A 70 3.782 −67.497 48.762 1.00 84.70 C ATOM 504 C GLY A 70 3.085 −66.235 49.284 1.00 85.99 C ATOM 505 O GLY A 70 1.851 −66.166 49.320 1.00 85.72 O ATOM 506 N LEU A 71 3.885 −65.232 49.669 1.00 79.59 N ATOM 507 CA LEU A 71 3.368 −63.971 50.216 1.00 77.19 C ATOM 508 C LEU A 71 3.142 −62.997 49.049 1.00 72.56 C ATOM 509 O LEU A 71 3.509 −63.311 47.915 1.00 69.12 O ATOM 510 CB LEU A 71 4.316 −63.344 51.275 1.00 79.65 C ATOM 511 CG LEU A 71 5.393 −64.243 51.941 1.00 87.68 C ATOM 512 CD1 LEU A 71 6.771 −63.964 51.355 1.00 87.45 C ATOM 513 CD2 LEU A 71 5.451 −64.021 53.438 1.00 93.95 C ATOM 514 N LEU A 72 2.537 −61.832 49.333 1.00 65.30 N ATOM 515 CA LEU A 72 2.279 −60.794 48.341 1.00 61.42 C ATOM 516 C LEU A 72 3.191 −59.606 48.602 1.00 63.54 C ATOM 517 O LEU A 72 3.126 −59.005 49.673 1.00 64.71 O ATOM 518 CB LEU A 72 0.814 −60.330 48.397 1.00 60.90 C ATOM 519 CG LEU A 72 −0.196 −61.290 47.770 1.00 64.83 C ATOM 520 CD1 LEU A 72 −1.363 −61.547 48.701 1.00 67.47 C ATOM 521 CD2 LEU A 72 −0.695 −60.772 46.446 1.00 61.31 C ATOM 522 N VAL A 73 4.035 −59.268 47.626 1.00 57.45 N ATOM 523 CA VAL A 73 4.911 −58.099 47.696 1.00 57.01 C ATOM 524 C VAL A 73 4.038 −56.887 47.373 1.00 55.93 C ATOM 525 O VAL A 73 4.194 −55.834 47.989 1.00 55.91 O ATOM 526 CB VAL A 73 6.101 −58.213 46.708 1.00 60.68 C ATOM 527 CG1 VAL A 73 6.954 −56.943 46.721 1.00 61.41 C ATOM 528 CG2 VAL A 73 6.955 −59.435 47.033 1.00 62.36 C ATOM 529 N ARG A 74 3.143 −57.041 46.376 1.00 48.50 N ATOM 530 CA ARG A 74 2.212 −56.004 45.944 1.00 45.91 C ATOM 531 C ARG A 74 0.851 −56.650 45.712 1.00 49.19 C ATOM 532 O ARG A 74 0.779 −57.679 45.044 1.00 48.25 O ATOM 533 CB ARG A 74 2.721 −55.363 44.646 1.00 42.04 C ATOM 534 CG ARG A 74 2.239 −53.942 44.375 1.00 48.38 C ATOM 535 CD ARG A 74 2.887 −53.406 43.099 1.00 51.48 C ATOM 536 NE ARG A 74 2.348 −54.060 41.899 1.00 44.28 N ATOM 537 CZ ARG A 74 3.001 −54.265 40.749 1.00 52.67 C ATOM 538 NH1 ARG A 74 4.269 −53.880 40.610 1.00 38.62 N ATOM 539 NH2 ARG A 74 2.394 −54.874 39.735 1.00 32.16 N ATOM 540 N ASN A 75 −0.221 −56.060 46.258 1.00 46.73 N ATOM 541 CA ASN A 75 −1.582 −56.565 46.042 1.00 46.00 C ATOM 542 C ASN A 75 −2.053 −56.170 44.652 1.00 46.93 C ATOM 543 O ASN A 75 −1.578 −55.172 44.105 1.00 44.44 O ATOM 544 CB ASN A 75 −2.563 −55.972 47.063 1.00 50.08 C ATOM 545 CG ASN A 75 −2.362 −56.476 48.469 1.00 78.19 C ATOM 546 OD1 ASN A 75 −2.311 −57.685 48.705 1.00 72.92 O ATOM 547 ND2 ASN A 75 −2.295 −55.574 49.446 1.00 75.87 N ATOM 548 N CYS A 76 −3.031 −56.913 44.113 1.00 43.83 N ATOM 549 CA CYS A 76 −3.631 −56.569 42.837 1.00 42.07 C ATOM 550 C CYS A 76 −4.596 −55.401 43.046 1.00 44.28 C ATOM 551 O CYS A 76 −5.402 −55.433 43.972 1.00 45.68 O ATOM 552 CB CYS A 76 −4.344 −57.754 42.188 1.00 42.61 C ATOM 553 SG CYS A 76 −4.559 −57.561 40.392 1.00 45.25 S ATOM 554 N THR A 77 −4.504 −54.385 42.184 1.00 38.93 N ATOM 555 CA THR A 77 −5.386 −53.218 42.158 1.00 37.81 C ATOM 556 C THR A 77 −6.042 −53.265 40.782 1.00 37.95 C ATOM 557 O THR A 77 −5.600 −54.044 39.940 1.00 34.24 O ATOM 558 CB THR A 77 −4.589 −51.912 42.354 1.00 47.00 C ATOM 559 OG1 THR A 77 −3.827 −51.635 41.183 1.00 52.34 O ATOM 560 CG2 THR A 77 −3.669 −51.957 43.564 1.00 48.24 C ATOM 561 N ILE A 78 −7.052 −52.423 40.526 1.00 34.74 N ATOM 562 CA ILE A 78 −7.727 −52.440 39.221 1.00 32.87 C ATOM 563 C ILE A 78 −6.832 −51.967 38.054 1.00 34.22 C ATOM 564 O ILE A 78 −7.246 −52.115 36.919 1.00 31.53 O ATOM 565 CB ILE A 78 −9.084 −51.696 39.222 1.00 35.74 C ATOM 566 CG1 ILE A 78 −8.952 −50.234 39.719 1.00 35.66 C ATOM 567 CG2 ILE A 78 −10.114 −52.489 40.041 1.00 36.24 C ATOM 568 CD1 ILE A 78 −10.064 −49.391 39.304 1.00 39.66 C ATOM 569 N THR A 79 −5.607 −51.459 38.309 1.00 31.55 N ATOM 570 CA THR A 79 −4.687 −51.066 37.244 1.00 30.47 C ATOM 571 C THR A 79 −3.338 −51.805 37.296 1.00 34.54 C ATOM 572 O THR A 79 −2.495 −51.553 36.439 1.00 35.67 O ATOM 573 CB THR A 79 −4.466 −49.536 37.282 1.00 35.81 C ATOM 574 OG1 THR A 79 −3.816 −49.184 38.504 1.00 37.07 O ATOM 575 CG2 THR A 79 −5.763 −48.750 37.150 1.00 32.44 C ATOM 576 N ALA A 80 −3.107 −52.680 38.277 1.00 30.96 N ATOM 577 CA ALA A 80 −1.819 −53.347 38.406 1.00 31.21 C ATOM 578 C ALA A 80 −1.958 −54.767 38.929 1.00 35.69 C ATOM 579 O ALA A 80 −2.756 −55.035 39.831 1.00 35.56 O ATOM 580 CB ALA A 80 −0.918 −52.540 39.332 1.00 32.83 C ATOM 581 N ASN A 81 −1.122 −55.665 38.395 1.00 31.83 N ATOM 582 CA ASN A 81 −1.123 −57.068 38.775 1.00 31.50 C ATOM 583 C ASN A 81 −0.544 −57.282 40.171 1.00 34.61 C ATOM 584 O ASN A 81 0.219 −56.452 40.677 1.00 33.14 O ATOM 585 CB ASN A 81 −0.322 −57.899 37.756 1.00 31.63 C ATOM 586 CG ASN A 81 −0.760 −59.340 37.647 1.00 35.72 C ATOM 587 OD1 ASN A 81 −1.474 −59.868 38.492 1.00 31.77 O ATOM 588 ND2 ASN A 81 −0.309 −60.026 36.625 1.00 31.52 N ATOM 589 N ALA A 82 −0.883 −58.425 40.777 1.00 32.98 N ATOM 590 CA ALA A 82 −0.312 −58.828 42.056 1.00 33.88 C ATOM 591 C ALA A 82 1.136 −59.211 41.803 1.00 36.40 C ATOM 592 O ALA A 82 1.468 −59.646 40.701 1.00 34.67 O ATOM 593 CB ALA A 82 −1.058 −60.032 42.630 1.00 35.42 C ATOM 594 N GLU A 83 1.997 −59.032 42.804 1.00 34.63 N ATOM 595 CA GLU A 83 3.398 −59.444 42.733 1.00 35.26 C ATOM 596 C GLU A 83 3.664 −60.360 43.922 1.00 45.22 C ATOM 597 O GLU A 83 3.365 −59.978 45.052 1.00 45.33 O ATOM 598 CB GLU A 83 4.335 −58.241 42.709 1.00 35.74 C ATOM 599 CG GLU A 83 4.312 −57.536 41.361 1.00 38.29 C ATOM 600 CD GLU A 83 4.982 −58.293 40.227 1.00 39.60 C ATOM 601 OE1 GLU A 83 6.020 −58.944 40.471 1.00 37.67 O ATOM 602 OE2 GLU A 83 4.464 −58.239 39.092 1.00 33.86 O ATOM 603 N CYS A 84 4.176 −61.580 43.661 1.00 46.59 N ATOM 604 CA CYS A 84 4.408 −62.600 44.689 1.00 50.51 C ATOM 605 C CYS A 84 5.857 −62.692 45.154 1.00 56.34 C ATOM 606 O CYS A 84 6.767 −62.144 44.535 1.00 54.93 O ATOM 607 CB CYS A 84 3.930 −63.970 44.209 1.00 52.50 C ATOM 608 SG CYS A 84 2.338 −63.963 43.346 1.00 55.78 S ATOM 609 N ALA A 85 6.051 −63.457 46.239 1.00 56.45 N ATOM 610 CA ALA A 85 7.354 −63.789 46.820 1.00 59.32 C ATOM 611 C ALA A 85 7.234 −65.102 47.595 1.00 67.61 C ATOM 612 O ALA A 85 6.138 −65.454 48.033 1.00 66.03 O ATOM 613 CB ALA A 85 7.829 −62.683 47.746 1.00 61.11 C ATOM 614 O CYS A 86 9.789 −65.864 50.035 1.00 80.95 O ATOM 615 N CYS A 86 8.352 −65.823 47.747 1.00 70.04 N ATOM 616 CA CYS A 86 8.406 −67.079 48.506 1.00 73.93 C ATOM 617 C CYS A 86 8.999 −66.800 49.883 1.00 81.58 C ATOM 618 CB CYS A 86 9.220 −68.131 47.758 1.00 75.75 C ATOM 619 SG CYS A 86 8.510 −68.637 46.167 1.00 77.80 S ATOM 620 O ARG A 87 10.966 −68.856 51.499 1.00 93.44 O ATOM 621 N ARG A 87 8.628 −67.615 50.882 1.00 82.66 N ATOM 622 CA ARG A 87 9.152 −67.478 52.247 1.00 86.62 C ATOM 623 C ARG A 87 10.609 −67.957 52.266 1.00 93.77 C ATOM 624 CB ARG A 87 8.311 −68.300 53.244 1.00 90.21 C ATOM 625 CG ARG A 87 6.862 −67.814 53.373 1.00 102.71 C ATOM 626 CD ARG A 87 5.855 −68.945 53.538 1.00 117.92 C ATOM 627 NE ARG A 87 4.501 −68.533 53.151 1.00 127.83 N ATOM 628 CZ ARG A 87 3.660 −67.820 53.902 1.00 144.91 C ATOM 629 NH1 ARG A 87 4.016 −67.412 55.118 1.00 137.73 N ATOM 630 NH2 ARG A 87 2.454 −67.506 53.440 1.00 128.40 N ATOM 631 O ASN A 88 12.123 −69.760 54.308 1.00 100.54 O ATOM 632 N ASN A 88 11.451 −67.357 53.130 1.00 92.99 N ATOM 633 CA ASN A 88 12.872 −67.726 53.238 1.00 95.23 C ATOM 634 C ASN A 88 13.063 −69.146 53.806 1.00 100.81 C ATOM 635 CB ASN A 88 13.632 −66.706 54.103 1.00 99.16 C ATOM 636 CG ASN A 88 15.124 −66.958 54.200 1.00 131.25 C ATOM 637 OD1 ASN A 88 15.760 −67.438 53.255 1.00 126.92 O ATOM 638 ND2 ASN A 88 15.729 −66.618 55.332 1.00 127.85 N ATOM 639 O ASP A 94 9.704 −67.692 39.489 1.00 73.11 O ATOM 640 N ASP A 94 10.269 −69.594 36.923 1.00 76.76 N ATOM 641 CA ASP A 94 9.121 −68.883 37.478 1.00 72.67 C ATOM 642 C ASP A 94 9.610 −67.653 38.260 1.00 72.46 C ATOM 643 CB ASP A 94 8.283 −69.836 38.368 1.00 75.16 C ATOM 644 CG ASP A 94 6.962 −69.267 38.867 1.00 83.13 C ATOM 645 OD1 ASP A 94 6.180 −68.756 38.033 1.00 82.79 O ATOM 646 OD2 ASP A 94 6.687 −69.383 40.082 1.00 85.88 O ATOM 647 O LYS A 95 9.445 −63.752 39.792 1.00 58.23 O ATOM 648 N LYS A 95 9.918 −66.548 37.542 1.00 64.62 N ATOM 649 CA LYS A 95 10.353 −65.294 38.179 1.00 61.76 C ATOM 650 C LYS A 95 9.199 −64.628 38.960 1.00 59.06 C ATOM 651 CB LYS A 95 10.930 −64.306 37.136 1.00 63.69 C ATOM 652 CG LYS A 95 9.877 −63.631 36.254 1.00 61.55 C ATOM 653 CD LYS A 95 10.458 −63.036 34.990 1.00 56.57 C ATOM 654 CE LYS A 95 9.357 −62.586 34.067 1.00 47.71 C ATOM 655 NZ LYS A 95 9.895 −61.924 32.850 1.00 44.34 N ATOM 656 N GLU A 96 7.946 −65.028 38.670 1.00 51.16 N ATOM 657 CA GLU A 96 6.767 −64.483 39.328 1.00 47.79 C ATOM 658 C GLU A 96 6.452 −65.102 40.698 1.00 53.73 C ATOM 659 O GLU A 96 5.500 −64.649 41.318 1.00 51.16 O ATOM 660 CB GLU A 96 5.550 −64.578 38.385 1.00 46.46 C ATOM 661 CG GLU A 96 5.723 −63.777 37.098 1.00 46.51 C ATOM 662 CD GLU A 96 5.931 −62.283 37.282 1.00 56.44 C ATOM 663 OE1 GLU A 96 5.605 −61.761 38.374 1.00 47.31 O ATOM 664 OE2 GLU A 96 6.408 −61.627 36.326 1.00 40.33 O ATOM 665 N CYS A 97 7.227 −66.111 41.186 1.00 54.71 N ATOM 666 CA CYS A 97 7.038 −66.714 42.517 1.00 57.34 C ATOM 667 C CYS A 97 5.582 −67.130 42.819 1.00 59.38 C ATOM 668 O CYS A 97 5.113 −66.975 43.946 1.00 58.58 O ATOM 669 CB CYS A 97 7.573 −65.776 43.594 1.00 59.57 C ATOM 670 SG CYS A 97 9.187 −65.050 43.208 1.00 65.12 S ATOM 671 N THR A 98 4.875 −67.628 41.798 1.00 56.33 N ATOM 672 CA THR A 98 3.497 −68.101 41.922 1.00 56.86 C ATOM 673 C THR A 98 3.531 −69.509 42.570 1.00 64.64 C ATOM 674 O THR A 98 2.565 −69.898 43.231 1.00 64.61 O ATOM 675 CB THR A 98 2.780 −68.010 40.550 1.00 67.68 C ATOM 676 OG1 THR A 98 2.314 −66.665 40.377 1.00 69.41 O ATOM 677 CG2 THR A 98 1.605 −68.975 40.412 1.00 69.92 C ATOM 678 N GLU A 99 4.659 −70.249 42.387 1.00 63.98 N ATOM 679 CA GLU A 99 4.902 −71.579 42.956 1.00 67.53 C ATOM 680 C GLU A 99 6.206 −71.525 43.771 1.00 74.19 C ATOM 681 O GLU A 99 7.172 −70.901 43.320 1.00 72.07 O ATOM 682 CB GLU A 99 5.051 −72.635 41.842 1.00 70.22 C ATOM 683 CG GLU A 99 3.904 −72.679 40.836 1.00 81.49 C ATOM 684 CD GLU A 99 4.178 −72.104 39.455 1.00 103.65 C ATOM 685 OE1 GLU A 99 5.323 −72.237 38.962 1.00 101.28 O ATOM 686 OE2 GLU A 99 3.223 −71.582 38.834 1.00 92.97 O ATOM 687 N CYS A 100 6.225 −72.166 44.967 1.00 74.67 N ATOM 688 CA CYS A 100 7.392 −72.212 45.860 1.00 77.43 C ATOM 689 C CYS A 100 7.622 −73.654 46.338 1.00 85.72 C ATOM 690 O CYS A 100 7.952 −74.538 45.551 1.00 86.74 O ATOM 691 CB CYS A 100 7.204 −71.267 47.048 1.00 77.96 C ATOM 692 SG CYS A 100 6.750 −69.568 46.600 1.00 78.35 S ATOM 693 CD CD A 9901 −7.490 −41.157 31.507 0.50 62.49 CD ATOM 694 CD CD A 9902 −6.163 −59.542 27.858 1.00 39.29 CD ATOM 695 N GLU B 1 −16.461 −26.648 36.434 1.00 66.26 N ATOM 696 CA GLU B 1 −16.304 −25.892 35.191 1.00 63.97 C ATOM 697 C GLU B 1 −14.864 −25.409 35.022 1.00 68.14 C ATOM 698 O GLU B 1 −14.252 −24.969 35.997 1.00 70.24 O ATOM 699 CB GLU B 1 −17.259 −24.686 35.173 1.00 64.42 C ATOM 700 CG GLU B 1 −17.296 −23.947 33.847 1.00 72.66 C ATOM 701 CD GLU B 1 −18.367 −22.878 33.729 1.00 92.40 C ATOM 702 OE1 GLU B 1 −19.570 −23.226 33.767 1.00 83.52 O ATOM 703 OE2 GLU B 1 −17.999 −21.696 33.539 1.00 84.58 O ATOM 704 N ILE B 2 −14.352 −25.433 33.770 1.00 62.46 N ATOM 705 CA ILE B 2 −13.013 −24.931 33.439 1.00 62.90 C ATOM 706 C ILE B 2 −13.223 −23.594 32.711 1.00 63.43 C ATOM 707 O ILE B 2 −13.939 −23.538 31.709 1.00 61.42 O ATOM 708 CB ILE B 2 −12.071 −25.936 32.686 1.00 66.24 C ATOM 709 CG1 ILE B 2 −11.426 −25.308 31.422 1.00 65.76 C ATOM 710 CG2 ILE B 2 −12.747 −27.276 32.367 1.00 66.51 C ATOM 711 CD1 ILE B 2 −10.366 −26.070 30.866 1.00 72.40 C ATOM 712 N VAL B 3 −12.613 −22.522 33.235 1.00 59.20 N ATOM 713 CA VAL B 3 −12.742 −21.176 32.690 1.00 57.38 C ATOM 714 C VAL B 3 −11.638 −20.926 31.664 1.00 59.89 C ATOM 715 O VAL B 3 −10.461 −21.101 31.983 1.00 61.71 O ATOM 716 CB VAL B 3 −12.712 −20.125 33.834 1.00 63.01 C ATOM 717 CG1 VAL B 3 −12.856 −18.704 33.291 1.00 62.18 C ATOM 718 CG2 VAL B 3 −13.804 −20.412 34.865 1.00 62.58 C ATOM 719 N LEU B 4 −12.020 −20.485 30.446 1.00 53.70 N ATOM 720 CA LEU B 4 −11.085 −20.140 29.376 1.00 53.06 C ATOM 721 C LEU B 4 −11.001 −18.612 29.315 1.00 58.51 C ATOM 722 O LEU B 4 −12.030 −17.950 29.155 1.00 57.19 O ATOM 723 CB LEU B 4 −11.559 −20.701 28.028 1.00 50.15 C ATOM 724 CG LEU B 4 −11.644 −22.224 27.914 1.00 52.38 C ATOM 725 CD1 LEU B 4 −12.192 −22.628 26.562 1.00 49.24 C ATOM 726 CD2 LEU B 4 −10.294 −22.869 28.132 1.00 56.14 C ATOM 727 N THR B 5 −9.787 −18.053 29.488 1.00 57.12 N ATOM 728 CA THR B 5 −9.545 −16.612 29.492 1.00 57.49 C ATOM 729 C THR B 5 −8.755 −16.226 28.244 1.00 62.49 C ATOM 730 O THR B 5 −7.591 −16.606 28.115 1.00 64.71 O ATOM 731 CB THR B 5 −8.829 −16.210 30.792 1.00 64.16 C ATOM 732 OG1 THR B 5 −9.615 −16.656 31.899 1.00 61.62 O ATOM 733 CG2 THR B 5 −8.622 −14.706 30.907 1.00 64.87 C ATOM 734 N GLN B 6 −9.392 −15.478 27.330 1.00 57.23 N ATOM 735 CA GLN B 6 −8.780 −15.010 26.088 1.00 57.57 C ATOM 736 C GLN B 6 −8.200 −13.609 26.227 1.00 65.40 C ATOM 737 O GLN B 6 −8.845 −12.745 26.822 1.00 65.16 O ATOM 738 CB GLN B 6 −9.805 −15.033 24.948 1.00 55.44 C ATOM 739 CG GLN B 6 −10.142 −16.440 24.518 1.00 58.07 C ATOM 740 CD GLN B 6 −11.047 −16.457 23.319 1.00 61.09 C ATOM 741 OE1 GLN B 6 −12.254 −16.645 23.435 1.00 50.30 O ATOM 742 NE2 GLN B 6 −10.491 −16.247 22.143 1.00 50.28 N ATOM 743 N SER B 7 −6.996 −13.377 25.655 1.00 64.70 N ATOM 744 CA SER B 7 −6.319 −12.075 25.708 1.00 66.73 C ATOM 745 C SER B 7 −5.802 −11.675 24.312 1.00 70.20 C ATOM 746 O SER B 7 −5.273 −12.531 23.601 1.00 69.56 O ATOM 747 CB SER B 7 −5.149 −12.106 26.693 1.00 73.95 C ATOM 748 OG SER B 7 −5.320 −13.078 27.711 1.00 85.76 O ATOM 749 N PRO B 8 −5.891 −10.382 23.933 1.00 67.08 N ATOM 750 CA PRO B 8 −6.555 −9.260 24.611 1.00 66.41 C ATOM 751 C PRO B 8 −8.047 −9.281 24.239 1.00 66.56 C ATOM 752 O PRO B 8 −8.455 −10.091 23.401 1.00 63.39 O ATOM 753 CB PRO B 8 −5.836 −8.040 24.029 1.00 69.44 C ATOM 754 CG PRO B 8 −5.503 −8.455 22.641 1.00 73.38 C ATOM 755 CD PRO B 8 −5.140 −9.917 22.751 1.00 69.21 C ATOM 756 N ALA B 9 −8.852 −8.374 24.826 1.00 62.75 N ATOM 757 CA ALA B 9 −10.283 −8.275 24.497 1.00 59.18 C ATOM 758 C ALA B 9 −10.422 −7.798 23.048 1.00 60.93 C ATOM 759 O ALA B 9 −11.145 −8.410 22.262 1.00 57.16 O ATOM 760 CB ALA B 9 −10.985 −7.309 25.443 1.00 59.92 C ATOM 761 N THR B 10 −9.679 −6.735 22.695 1.00 59.34 N ATOM 762 CA THR B 10 −9.622 −6.197 21.336 1.00 58.27 C ATOM 763 C THR B 10 −8.158 −6.209 20.896 1.00 63.97 C ATOM 764 O THR B 10 −7.299 −5.732 21.640 1.00 66.81 O ATOM 765 CB THR B 10 −10.212 −4.783 21.288 1.00 65.78 C ATOM 766 OG1 THR B 10 −11.542 −4.832 21.810 1.00 64.14 O ATOM 767 CG2 THR B 10 −10.233 −4.195 19.868 1.00 62.11 C ATOM 768 N LEU B 11 −7.874 −6.767 19.709 1.00 58.45 N ATOM 769 CA LEU B 11 −6.526 −6.820 19.147 1.00 59.88 C ATOM 770 C LEU B 11 −6.550 −5.961 17.892 1.00 63.08 C ATOM 771 O LEU B 11 −7.034 −6.403 16.848 1.00 61.01 O ATOM 772 CB LEU B 11 −6.113 −8.276 18.833 1.00 59.83 C ATOM 773 CG LEU B 11 −4.713 −8.487 18.236 1.00 66.84 C ATOM 774 CD1 LEU B 11 −3.620 −7.868 19.113 1.00 69.57 C ATOM 775 CD2 LEU B 11 −4.425 −9.967 18.049 1.00 68.76 C ATOM 776 N SER B 12 −6.094 −4.700 18.022 1.00 60.82 N ATOM 777 CA SER B 12 −6.085 −3.725 16.931 1.00 59.54 C ATOM 778 C SER B 12 −4.750 −3.781 16.181 1.00 66.36 C ATOM 779 O SER B 12 −3.742 −3.296 16.695 1.00 68.95 O ATOM 780 CB SER B 12 −6.343 −2.321 17.473 1.00 61.03 C ATOM 781 OG SER B 12 −7.545 −2.264 18.225 1.00 59.35 O ATOM 782 N LEU B 13 −4.742 −4.389 14.972 1.00 61.99 N ATOM 783 CA LEU B 13 −3.552 −4.515 14.115 1.00 64.21 C ATOM 784 C LEU B 13 −3.915 −4.162 12.663 1.00 68.04 C ATOM 785 O LEU B 13 −5.080 −4.297 12.288 1.00 63.88 O ATOM 786 CB LEU B 13 −3.008 −5.948 14.169 1.00 64.67 C ATOM 787 CG LEU B 13 −2.533 −6.450 15.532 1.00 70.50 C ATOM 788 CD1 LEU B 13 −2.338 −7.955 15.506 1.00 69.97 C ATOM 789 CD2 LEU B 13 −1.250 −5.748 15.966 1.00 76.02 C ATOM 790 N SER B 14 −2.939 −3.681 11.859 1.00 68.35 N ATOM 791 CA SER B 14 −3.173 −3.305 10.453 1.00 68.36 C ATOM 792 C SER B 14 −3.015 −4.520 9.513 1.00 72.45 C ATOM 793 O SER B 14 −2.397 −5.504 9.921 1.00 72.71 O ATOM 794 CB SER B 14 −2.176 −2.235 10.012 1.00 76.14 C ATOM 795 OG SER B 14 −2.104 −1.139 10.908 1.00 88.84 O ATOM 796 N PRO B 15 −3.510 −4.442 8.241 1.00 69.11 N ATOM 797 CA PRO B 15 −3.303 −5.542 7.286 1.00 69.65 C ATOM 798 C PRO B 15 −1.819 −5.655 6.930 1.00 78.54 C ATOM 799 O PRO B 15 −1.171 −4.641 6.663 1.00 80.10 O ATOM 800 CB PRO B 15 −4.158 −5.144 6.079 1.00 69.78 C ATOM 801 CG PRO B 15 −5.220 −4.292 6.634 1.00 71.64 C ATOM 802 CD PRO B 15 −4.601 −3.552 7.789 1.00 68.61 C ATOM 803 N GLY B 16 −1.278 −6.878 6.977 1.00 77.14 N ATOM 804 CA GLY B 16 0.134 −7.142 6.736 1.00 80.94 C ATOM 805 C GLY B 16 0.838 −7.494 8.053 1.00 86.59 C ATOM 806 O GLY B 16 1.821 −8.232 8.019 1.00 89.09 O ATOM 807 N GLU B 17 0.353 −6.967 9.209 1.00 81.49 N ATOM 808 CA GLU B 17 0.953 −7.263 10.516 1.00 82.40 C ATOM 809 C GLU B 17 0.663 −8.702 10.959 1.00 84.44 C ATOM 810 O GLU B 17 −0.209 −9.370 10.401 1.00 81.35 O ATOM 811 CB GLU B 17 0.466 −6.282 11.606 1.00 82.63 C ATOM 812 CG GLU B 17 0.950 −4.853 11.438 1.00 93.06 C ATOM 813 CD GLU B 17 0.713 −3.988 12.663 1.00 110.22 C ATOM 814 OE1 GLU B 17 1.356 −4.242 13.708 1.00 100.30 O ATOM 815 OE2 GLU B 17 −0.138 −3.072 12.588 1.00 101.98 O ATOM 816 N ARG B 18 1.409 −9.166 11.968 1.00 82.58 N ATOM 817 CA ARG B 18 1.280 −10.506 12.533 1.00 81.15 C ATOM 818 C ARG B 18 0.391 −10.433 13.778 1.00 82.43 C ATOM 819 O ARG B 18 0.608 −9.564 14.625 1.00 83.25 O ATOM 820 CB ARG B 18 2.674 −11.074 12.872 1.00 83.89 C ATOM 821 CG ARG B 18 2.660 −12.393 13.638 1.00 94.10 C ATOM 822 CD ARG B 18 3.789 −13.313 13.217 1.00 105.86 C ATOM 823 NE ARG B 18 3.869 −14.499 14.073 1.00 109.81 N ATOM 824 CZ ARG B 18 4.557 −15.605 13.794 1.00 126.65 C ATOM 825 NH1 ARG B 18 5.243 −15.703 12.657 1.00 120.71 N ATOM 826 NH2 ARG B 18 4.561 −16.624 14.644 1.00 112.23 N ATOM 827 N ALA B 19 −0.605 −11.342 13.887 1.00 74.95 N ATOM 828 CA ALA B 19 −1.521 −11.397 15.031 1.00 72.03 C ATOM 829 C ALA B 19 −1.317 −12.678 15.822 1.00 75.61 C ATOM 830 O ALA B 19 −1.129 −13.737 15.230 1.00 74.37 O ATOM 831 CB ALA B 19 −2.959 −11.320 14.555 1.00 69.03 C ATOM 832 N THR B 20 −1.358 −12.573 17.157 1.00 72.69 N ATOM 833 CA THR B 20 −1.212 −13.702 18.069 1.00 73.06 C ATOM 834 C THR B 20 −2.342 −13.556 19.104 1.00 74.31 C ATOM 835 O THR B 20 −2.355 −12.600 19.882 1.00 73.95 O ATOM 836 CB THR B 20 0.265 −13.789 18.581 1.00 90.30 C ATOM 837 OG1 THR B 20 0.843 −15.022 18.139 1.00 92.46 O ATOM 838 CG2 THR B 20 0.417 −13.666 20.106 1.00 91.87 C ATOM 839 N LEU B 21 −3.350 −14.446 19.016 1.00 68.30 N ATOM 840 CA LEU B 21 −4.521 −14.459 19.896 1.00 65.65 C ATOM 841 C LEU B 21 −4.298 −15.577 20.900 1.00 68.48 C ATOM 842 O LEU B 21 −4.028 −16.695 20.480 1.00 68.53 O ATOM 843 CB LEU B 21 −5.800 −14.732 19.087 1.00 62.61 C ATOM 844 CG LEU B 21 −6.278 −13.614 18.155 1.00 66.57 C ATOM 845 CD1 LEU B 21 −5.452 −13.557 16.865 1.00 68.10 C ATOM 846 CD2 LEU B 21 −7.744 −13.808 17.793 1.00 66.13 C ATOM 847 N SER B 22 −4.389 −15.288 22.207 1.00 64.91 N ATOM 848 CA SER B 22 −4.132 −16.278 23.255 1.00 66.06 C ATOM 849 C SER B 22 −5.391 −16.739 23.990 1.00 67.57 C ATOM 850 O SER B 22 −6.364 −15.995 24.088 1.00 64.89 O ATOM 851 CB SER B 22 −3.117 −15.733 24.257 1.00 74.58 C ATOM 852 OG SER B 22 −3.684 −14.816 25.180 1.00 85.87 O ATOM 853 N CYS B 23 −5.343 −17.978 24.513 1.00 64.28 N ATOM 854 CA CYS B 23 −6.405 −18.615 25.301 1.00 62.53 C ATOM 855 C CYS B 23 −5.697 −19.297 26.483 1.00 68.85 C ATOM 856 O CYS B 23 −4.734 −20.036 26.275 1.00 69.65 O ATOM 857 CB CYS B 23 −7.191 −19.618 24.451 1.00 60.39 C ATOM 858 SG CYS B 23 −8.582 −20.425 25.303 1.00 62.06 S ATOM 859 N SER B 24 −6.126 −18.989 27.714 1.00 66.17 N ATOM 860 CA SER B 24 −5.555 −19.540 28.941 1.00 67.93 C ATOM 861 C SER B 24 −6.639 −20.319 29.684 1.00 69.58 C ATOM 862 O SER B 24 −7.686 −19.750 29.989 1.00 67.28 O ATOM 863 CB SER B 24 −5.025 −18.413 29.824 1.00 73.52 C ATOM 864 OG SER B 24 −4.551 −18.904 31.068 1.00 85.22 O ATOM 865 N ALA B 25 −6.384 −21.604 29.994 1.00 66.33 N ATOM 866 CA ALA B 25 −7.331 −22.468 30.711 1.00 64.71 C ATOM 867 C ALA B 25 −7.006 −22.520 32.207 1.00 72.28 C ATOM 868 O ALA B 25 −5.835 −22.434 32.579 1.00 75.00 O ATOM 869 CB ALA B 25 −7.293 −23.866 30.128 1.00 64.17 C ATOM 870 N SER B 26 −8.036 −22.653 33.067 1.00 68.25 N ATOM 871 CA SER B 26 −7.843 −22.730 34.522 1.00 70.50 C ATOM 872 C SER B 26 −7.155 −24.046 34.917 1.00 76.92 C ATOM 873 O SER B 26 −6.318 −24.047 35.823 1.00 79.16 O ATOM 874 CB SER B 26 −9.172 −22.576 35.254 1.00 72.51 C ATOM 875 OG SER B 26 −10.107 −23.553 34.832 1.00 78.97 O ATOM 876 N SER B 27 −7.502 −25.155 34.230 1.00 72.77 N ATOM 877 CA SER B 27 −6.878 −26.473 34.408 1.00 73.84 C ATOM 878 C SER B 27 −6.429 −26.995 33.028 1.00 76.35 C ATOM 879 O SER B 27 −6.773 −26.393 32.009 1.00 74.05 O ATOM 880 CB SER B 27 −7.835 −27.446 35.093 1.00 76.56 C ATOM 881 OG SER B 27 −9.062 −27.576 34.395 1.00 84.78 O ATOM 882 N SER B 28 −5.640 −28.080 32.996 1.00 73.77 N ATOM 883 CA SER B 28 −5.076 −28.614 31.747 1.00 72.82 C ATOM 884 C SER B 28 −6.100 −29.091 30.694 1.00 72.96 C ATOM 885 O SER B 28 −7.062 −29.774 31.031 1.00 70.71 O ATOM 886 CB SER B 28 −4.096 −29.744 32.047 1.00 77.29 C ATOM 887 OG SER B 28 −3.024 −29.279 32.849 1.00 89.12 O ATOM 888 N VAL B 29 −5.851 −28.753 29.412 1.00 68.69 N ATOM 889 CA VAL B 29 −6.670 −29.146 28.253 1.00 66.05 C ATOM 890 C VAL B 29 −5.746 −29.659 27.149 1.00 71.82 C ATOM 891 O VAL B 29 −4.564 −29.321 27.145 1.00 73.88 O ATOM 892 CB VAL B 29 −7.616 −28.027 27.741 1.00 67.49 C ATOM 893 CG1 VAL B 29 −8.763 −27.814 28.707 1.00 66.23 C ATOM 894 CG2 VAL B 29 −6.877 −26.722 27.483 1.00 68.50 C ATOM 895 N SER B 30 −6.287 −30.448 26.206 1.00 67.52 N ATOM 896 CA SER B 30 −5.499 −31.109 25.163 1.00 68.55 C ATOM 897 C SER B 30 −5.754 −30.627 23.725 1.00 71.50 C ATOM 898 O SER B 30 −4.840 −30.063 23.114 1.00 75.11 O ATOM 899 CB SER B 30 −5.713 −32.621 25.242 1.00 71.25 C ATOM 900 OG SER B 30 −5.577 −33.110 26.567 1.00 85.11 O ATOM 901 N TYR B 31 −6.952 −30.888 23.173 1.00 61.50 N ATOM 902 CA TYR B 31 −7.275 −30.609 21.771 1.00 59.42 C ATOM 903 C TYR B 31 −8.187 −29.371 21.642 1.00 61.50 C ATOM 904 O TYR B 31 −9.401 −29.497 21.460 1.00 58.79 O ATOM 905 CB TYR B 31 −7.920 −31.868 21.154 1.00 59.05 C ATOM 906 CG TYR B 31 −6.937 −32.971 20.818 1.00 61.39 C ATOM 907 CD1 TYR B 31 −6.432 −33.807 21.801 1.00 64.53 C ATOM 908 CD2 TYR B 31 −6.607 −33.251 19.499 1.00 61.86 C ATOM 909 CE1 TYR B 31 −5.566 −34.853 21.491 1.00 66.11 C ATOM 910 CE2 TYR B 31 −5.743 −34.293 19.173 1.00 63.96 C ATOM 911 CZ TYR B 31 −5.206 −35.080 20.174 1.00 71.35 C ATOM 912 OH TYR B 31 −4.366 −36.127 19.869 1.00 73.45 O ATOM 913 N MET B 32 −7.585 −28.171 21.737 1.00 58.11 N ATOM 914 CA MET B 32 −8.303 −26.888 21.710 1.00 55.98 C ATOM 915 C MET B 32 −8.949 −26.559 20.350 1.00 55.87 C ATOM 916 O MET B 32 −8.293 −26.666 19.315 1.00 55.56 O ATOM 917 CB MET B 32 −7.343 −25.753 22.121 1.00 60.50 C ATOM 918 CG MET B 32 −8.010 −24.403 22.364 1.00 63.40 C ATOM 919 SD MET B 32 −9.069 −24.377 23.829 1.00 66.79 S ATOM 920 CE MET B 32 −7.881 −24.105 25.096 1.00 66.47 C ATOM 921 N HIS B 33 −10.226 −26.122 20.372 1.00 48.77 N ATOM 922 CA HIS B 33 −10.974 −25.722 19.175 1.00 46.33 C ATOM 923 C HIS B 33 −11.124 −24.210 19.150 1.00 49.53 C ATOM 924 O HIS B 33 −11.194 −23.588 20.208 1.00 49.20 O ATOM 925 CB HIS B 33 −12.366 −26.366 19.146 1.00 44.26 C ATOM 926 CG HIS B 33 −12.366 −27.863 19.196 1.00 46.92 C ATOM 927 ND1 HIS B 33 −11.430 −28.613 18.506 1.00 49.65 N ATOM 928 CD2 HIS B 33 −13.240 −28.702 19.793 1.00 47.01 C ATOM 929 CE1 HIS B 33 −11.742 −29.878 18.740 1.00 48.36 C ATOM 930 NE2 HIS B 33 −12.830 −29.978 19.497 1.00 47.24 N ATOM 931 N TRP B 34 −11.168 −23.625 17.944 1.00 45.71 N ATOM 932 CA TRP B 34 −11.312 −22.185 17.728 1.00 44.73 C ATOM 933 C TRP B 34 −12.417 −21.920 16.710 1.00 47.48 C ATOM 934 O TRP B 34 −12.510 −22.632 15.703 1.00 46.46 O ATOM 935 CB TRP B 34 −10.004 −21.565 17.235 1.00 45.07 C ATOM 936 CG TRP B 34 −8.893 −21.604 18.238 1.00 47.99 C ATOM 937 CD1 TRP B 34 −8.023 −22.631 18.461 1.00 52.28 C ATOM 938 CD2 TRP B 34 −8.451 −20.517 19.059 1.00 49.06 C ATOM 939 NE1 TRP B 34 −7.091 −22.266 19.408 1.00 53.74 N ATOM 940 CE2 TRP B 34 −7.335 −20.974 19.797 1.00 55.33 C ATOM 941 CE3 TRP B 34 −8.917 −19.212 19.277 1.00 49.76 C ATOM 942 CZ2 TRP B 34 −6.675 −20.166 20.728 1.00 56.59 C ATOM 943 CZ3 TRP B 34 −8.246 −18.404 20.181 1.00 53.00 C ATOM 944 CH2 TRP B 34 −7.133 −18.877 20.887 1.00 55.99 C ATOM 945 N TYR B 35 −13.254 −20.894 16.975 1.00 42.77 N ATOM 946 CA TYR B 35 −14.364 −20.516 16.103 1.00 40.95 C ATOM 947 C TYR B 35 −14.249 −19.067 15.695 1.00 44.85 C ATOM 948 O TYR B 35 −13.894 −18.229 16.516 1.00 44.61 O ATOM 949 CB TYR B 35 −15.717 −20.727 16.809 1.00 39.67 C ATOM 950 CG TYR B 35 −15.913 −22.159 17.231 1.00 40.42 C ATOM 951 CD1 TYR B 35 −16.385 −23.110 16.334 1.00 41.22 C ATOM 952 CD2 TYR B 35 −15.507 −22.594 18.486 1.00 41.05 C ATOM 953 CE1 TYR B 35 −16.428 −24.458 16.668 1.00 41.70 C ATOM 954 CE2 TYR B 35 −15.600 −23.932 18.850 1.00 41.03 C ATOM 955 CZ TYR B 35 −16.067 −24.863 17.939 1.00 46.65 C ATOM 956 OH TYR B 35 −16.164 −26.191 18.274 1.00 46.99 O ATOM 957 N GLN B 36 −14.589 −18.767 14.439 1.00 41.80 N ATOM 958 CA GLN B 36 −14.605 −17.397 13.930 1.00 41.65 C ATOM 959 C GLN B 36 −16.048 −16.955 13.872 1.00 43.53 C ATOM 960 O GLN B 36 −16.882 −17.731 13.413 1.00 42.02 O ATOM 961 CB GLN B 36 −14.006 −17.316 12.516 1.00 43.16 C ATOM 962 CG GLN B 36 −13.890 −15.873 11.994 1.00 46.31 C ATOM 963 CD GLN B 36 −13.693 −15.826 10.500 1.00 54.64 C ATOM 964 OE1 GLN B 36 −14.636 −16.029 9.731 1.00 47.51 O ATOM 965 NE2 GLN B 36 −12.468 −15.577 10.046 1.00 43.33 N ATOM 966 N GLN B 37 −16.342 −15.710 14.293 1.00 39.68 N ATOM 967 CA GLN B 37 −17.684 −15.146 14.187 1.00 38.25 C ATOM 968 C GLN B 37 −17.618 −13.721 13.644 1.00 43.10 C ATOM 969 O GLN B 37 −16.907 −12.885 14.206 1.00 42.94 O ATOM 970 CB GLN B 37 −18.438 −15.148 15.526 1.00 38.79 C ATOM 971 CG GLN B 37 −19.927 −14.852 15.318 1.00 41.09 C ATOM 972 CD GLN B 37 −20.759 −14.844 16.568 1.00 43.99 C ATOM 973 OE1 GLN B 37 −20.293 −14.519 17.659 1.00 38.85 O ATOM 974 NE2 GLN B 37 −22.047 −15.106 16.411 1.00 35.28 N ATOM 975 N LYS B 38 −18.410 −13.433 12.592 1.00 39.87 N ATOM 976 CA LYS B 38 −18.518 −12.090 12.009 1.00 39.92 C ATOM 977 C LYS B 38 −19.883 −11.515 12.408 1.00 43.53 C ATOM 978 O LYS B 38 −20.780 −12.304 12.713 1.00 40.65 O ATOM 979 CB LYS B 38 −18.331 −12.142 10.484 1.00 42.03 C ATOM 980 CG LYS B 38 −16.943 −12.663 10.102 1.00 46.18 C ATOM 981 CD LYS B 38 −16.493 −12.203 8.731 1.00 50.59 C ATOM 982 CE LYS B 38 −15.021 −12.437 8.512 1.00 55.26 C ATOM 983 NZ LYS B 38 −14.685 −12.470 7.065 1.00 61.75 N ATOM 984 N PRO B 39 −20.043 −10.154 12.456 1.00 42.90 N ATOM 985 CA PRO B 39 −21.298 −9.524 12.922 1.00 42.12 C ATOM 986 C PRO B 39 −22.591 −10.034 12.267 1.00 44.27 C ATOM 987 O PRO B 39 −22.654 −10.162 11.045 1.00 43.72 O ATOM 988 CB PRO B 39 −21.072 −8.028 12.646 1.00 44.44 C ATOM 989 CG PRO B 39 −19.604 −7.869 12.564 1.00 49.12 C ATOM 990 CD PRO B 39 −19.107 −9.129 11.945 1.00 44.70 C ATOM 991 N GLY B 40 −23.605 −10.365 13.100 1.00 40.97 N ATOM 992 CA GLY B 40 −24.905 −10.868 12.646 1.00 39.89 C ATOM 993 C GLY B 40 −24.868 −12.255 11.987 1.00 42.71 C ATOM 994 O GLY B 40 −25.853 −12.642 11.357 1.00 43.22 O ATOM 995 N GLN B 41 −23.761 −13.011 12.134 1.00 37.37 N ATOM 996 CA GLN B 41 −23.636 −14.344 11.548 1.00 36.16 C ATOM 997 C GLN B 41 −23.334 −15.355 12.642 1.00 36.54 C ATOM 998 O GLN B 41 −22.759 −15.013 13.672 1.00 36.09 O ATOM 999 CB GLN B 41 −22.513 −14.381 10.502 1.00 38.18 C ATOM 1000 CG GLN B 41 −22.705 −13.413 9.344 1.00 52.04 C ATOM 1001 CD GLN B 41 −21.473 −13.371 8.470 1.00 77.06 C ATOM 1002 OE1 GLN B 41 −20.890 −14.409 8.134 1.00 76.18 O ATOM 1003 NE2 GLN B 41 −21.050 −12.177 8.064 1.00 70.40 N ATOM 1004 N ALA B 42 −23.691 −16.610 12.393 1.00 32.84 N ATOM 1005 CA ALA B 42 −23.420 −17.710 13.315 1.00 31.84 C ATOM 1006 C ALA B 42 −21.902 −17.981 13.369 1.00 35.92 C ATOM 1007 O ALA B 42 −21.218 −17.679 12.384 1.00 35.98 O ATOM 1008 CB ALA B 42 −24.142 −18.971 12.841 1.00 31.79 C ATOM 1009 N PRO B 43 −21.372 −18.578 14.477 1.00 32.48 N ATOM 1010 CA PRO B 43 −19.945 −18.949 14.502 1.00 33.86 C ATOM 1011 C PRO B 43 −19.633 −20.046 13.442 1.00 38.94 C ATOM 1012 O PRO B 43 −20.540 −20.726 12.955 1.00 38.14 O ATOM 1013 CB PRO B 43 −19.721 −19.477 15.931 1.00 35.40 C ATOM 1014 CG PRO B 43 −20.847 −18.936 16.727 1.00 38.52 C ATOM 1015 CD PRO B 43 −22.007 −18.816 15.789 1.00 32.73 C ATOM 1016 N LYS B 44 −18.356 −20.181 13.086 1.00 36.90 N ATOM 1017 CA LYS B 44 −17.843 −21.142 12.105 1.00 37.55 C ATOM 1018 C LYS B 44 −16.636 −21.815 12.714 1.00 41.70 C ATOM 1019 O LYS B 44 −15.783 −21.104 13.259 1.00 40.89 O ATOM 1020 CB LYS B 44 −17.321 −20.391 10.853 1.00 42.52 C ATOM 1021 CG LYS B 44 −18.255 −20.311 9.655 1.00 54.87 C ATOM 1022 CD LYS B 44 −17.550 −19.585 8.497 1.00 57.24 C ATOM 1023 CE LYS B 44 −18.292 −19.641 7.180 1.00 62.53 C ATOM 1024 NZ LYS B 44 −19.476 −18.739 7.152 1.00 62.92 N ATOM 1025 N ARG B 45 −16.496 −23.156 12.558 1.00 37.53 N ATOM 1026 CA ARG B 45 −15.287 −23.856 13.001 1.00 37.89 C ATOM 1027 C ARG B 45 −14.124 −23.303 12.160 1.00 43.62 C ATOM 1028 O ARG B 45 −14.270 −23.152 10.947 1.00 42.49 O ATOM 1029 CB ARG B 45 −15.439 −25.376 12.836 1.00 35.88 C ATOM 1030 CG ARG B 45 −14.189 −26.180 13.215 1.00 38.81 C ATOM 1031 CD ARG B 45 −14.300 −27.660 12.899 1.00 44.81 C ATOM 1032 NE ARG B 45 −14.284 −27.916 11.454 1.00 49.62 N ATOM 1033 CZ ARG B 45 −14.013 −29.087 10.877 1.00 57.98 C ATOM 1034 NH1 ARG B 45 −13.730 −30.155 11.616 1.00 46.92 N ATOM 1035 NH2 ARG B 45 −14.012 −29.196 9.553 1.00 41.99 N ATOM 1036 N TRP B 46 −13.019 −22.914 12.817 1.00 43.32 N ATOM 1037 CA TRP B 46 −11.867 −22.321 12.136 1.00 45.71 C ATOM 1038 C TRP B 46 −10.631 −23.197 12.294 1.00 51.80 C ATOM 1039 O TRP B 46 −10.094 −23.677 11.295 1.00 52.82 O ATOM 1040 CB TRP B 46 −11.630 −20.887 12.650 1.00 44.76 C ATOM 1041 CG TRP B 46 −11.049 −20.002 11.603 1.00 47.19 C ATOM 1042 CD1 TRP B 46 −9.741 −19.642 11.470 1.00 52.39 C ATOM 1043 CD2 TRP B 46 −11.719 −19.516 10.435 1.00 46.66 C ATOM 1044 NE1 TRP B 46 −9.579 −18.869 10.345 1.00 52.63 N ATOM 1045 CE2 TRP B 46 −10.771 −18.793 9.678 1.00 52.22 C ATOM 1046 CE3 TRP B 46 −13.036 −19.601 9.963 1.00 46.11 C ATOM 1047 CZ2 TRP B 46 −11.093 −18.168 8.475 1.00 51.50 C ATOM 1048 CZ3 TRP B 46 −13.364 −18.952 8.784 1.00 47.71 C ATOM 1049 CH2 TRP B 46 −12.398 −18.246 8.053 1.00 50.27 C ATOM 1050 N ILE B 47 −10.195 −23.423 13.537 1.00 48.59 N ATOM 1051 CA ILE B 47 −9.077 −24.311 13.847 1.00 49.89 C ATOM 1052 C ILE B 47 −9.625 −25.312 14.846 1.00 53.75 C ATOM 1053 O ILE B 47 −10.351 −24.923 15.755 1.00 51.44 O ATOM 1054 CB ILE B 47 −7.819 −23.574 14.389 1.00 54.90 C ATOM 1055 CG1 ILE B 47 −7.425 −22.418 13.442 1.00 55.55 C ATOM 1056 CG2 ILE B 47 −6.641 −24.564 14.588 1.00 57.33 C ATOM 1057 CD1 ILE B 47 −6.154 −21.702 13.785 1.00 60.87 C ATOM 1058 N TYR B 48 −9.314 −26.598 14.661 1.00 52.51 N ATOM 1059 CA TYR B 48 −9.753 −27.659 15.568 1.00 51.57 C ATOM 1060 C TYR B 48 −8.586 −28.584 15.856 1.00 55.31 C ATOM 1061 O TYR B 48 −7.604 −28.578 15.116 1.00 55.78 O ATOM 1062 CB TYR B 48 −10.966 −28.422 15.011 1.00 51.70 C ATOM 1063 CG TYR B 48 −10.680 −29.256 13.781 1.00 55.49 C ATOM 1064 CD1 TYR B 48 −10.538 −28.669 12.530 1.00 58.03 C ATOM 1065 CD2 TYR B 48 −10.578 −30.642 13.867 1.00 56.79 C ATOM 1066 CE1 TYR B 48 −10.249 −29.431 11.400 1.00 59.64 C ATOM 1067 CE2 TYR B 48 −10.309 −31.417 12.741 1.00 58.73 C ATOM 1068 CZ TYR B 48 −10.143 −30.808 11.507 1.00 66.61 C ATOM 1069 OH TYR B 48 −9.858 −31.552 10.383 1.00 69.66 O ATOM 1070 N ASP B 49 −8.663 −29.323 16.971 1.00 51.22 N ATOM 1071 CA ASP B 49 −7.619 −30.252 17.411 1.00 52.44 C ATOM 1072 C ASP B 49 −6.282 −29.523 17.618 1.00 57.54 C ATOM 1073 O ASP B 49 −5.219 −30.047 17.279 1.00 57.96 O ATOM 1074 CB ASP B 49 −7.500 −31.454 16.434 1.00 54.46 C ATOM 1075 CG ASP B 49 −8.690 −32.406 16.448 1.00 59.93 C ATOM 1076 OD1 ASP B 49 −9.538 −32.292 17.359 1.00 57.16 O ATOM 1077 OD2 ASP B 49 −8.763 −33.282 15.561 1.00 66.68 O ATOM 1078 N THR B 50 −6.361 −28.279 18.153 1.00 54.10 N ATOM 1079 CA THR B 50 −5.232 −27.382 18.429 1.00 56.18 C ATOM 1080 C THR B 50 −4.659 −26.738 17.175 1.00 61.25 C ATOM 1081 O THR B 50 −4.566 −25.518 17.142 1.00 60.88 O ATOM 1082 CB THR B 50 −4.126 −28.075 19.260 1.00 62.52 C ATOM 1083 OG1 THR B 50 −4.720 −28.630 20.430 1.00 62.24 O ATOM 1084 CG2 THR B 50 −3.006 −27.126 19.665 1.00 59.52 C ATOM 1085 N SER B 51 −4.217 −27.545 16.185 1.00 60.20 N ATOM 1086 CA SER B 51 −3.523 −27.087 14.978 1.00 61.75 C ATOM 1087 C SER B 51 −4.168 −27.443 13.624 1.00 65.82 C ATOM 1088 O SER B 51 −3.609 −27.037 12.607 1.00 68.03 O ATOM 1089 CB SER B 51 −2.103 −27.651 14.998 1.00 66.64 C ATOM 1090 OG SER B 51 −2.114 −29.070 14.985 1.00 71.98 O ATOM 1091 N LYS B 52 −5.289 −28.194 13.568 1.00 59.53 N ATOM 1092 CA LYS B 52 −5.870 −28.542 12.264 1.00 58.69 C ATOM 1093 C LYS B 52 −6.749 −27.413 11.740 1.00 63.24 C ATOM 1094 O LYS B 52 −7.645 −26.962 12.448 1.00 61.69 O ATOM 1095 CB LYS B 52 −6.664 −29.858 12.312 1.00 58.90 C ATOM 1096 CG LYS B 52 −5.853 −31.056 12.784 1.00 67.94 C ATOM 1097 CD LYS B 52 −6.698 −32.319 12.753 1.00 73.07 C ATOM 1098 CE LYS B 52 −6.055 −33.486 13.455 1.00 79.98 C ATOM 1099 NZ LYS B 52 −7.006 −34.620 13.605 1.00 87.87 N ATOM 1100 N LEU B 53 −6.516 −26.974 10.495 1.00 62.00 N ATOM 1101 CA LEU B 53 −7.309 −25.909 9.878 1.00 61.40 C ATOM 1102 C LEU B 53 −8.542 −26.506 9.203 1.00 64.71 C ATOM 1103 O LEU B 53 −8.455 −27.582 8.605 1.00 65.02 O ATOM 1104 CB LEU B 53 −6.482 −25.145 8.830 1.00 63.57 C ATOM 1105 CG LEU B 53 −5.140 −24.583 9.275 1.00 70.86 C ATOM 1106 CD1 LEU B 53 −4.452 −23.882 8.121 1.00 72.90 C ATOM 1107 CD2 LEU B 53 −5.298 −23.627 10.446 1.00 72.79 C ATOM 1108 N ALA B 54 −9.689 −25.810 9.284 1.00 59.69 N ATOM 1109 CA ALA B 54 −10.917 −26.261 8.621 1.00 58.17 C ATOM 1110 C ALA B 54 −10.790 −26.015 7.104 1.00 64.70 C ATOM 1111 O ALA B 54 −9.942 −25.221 6.684 1.00 64.60 O ATOM 1112 CB ALA B 54 −12.119 −25.517 9.173 1.00 56.50 C ATOM 1113 N SER B 55 −11.607 −26.702 6.285 1.00 62.64 N ATOM 1114 CA SER B 55 −11.539 −26.554 4.822 1.00 64.02 C ATOM 1115 C SER B 55 −11.875 −25.116 4.396 1.00 68.19 C ATOM 1116 O SER B 55 −12.873 −24.546 4.854 1.00 66.10 O ATOM 1117 CB SER B 55 −12.466 −27.547 4.127 1.00 67.64 C ATOM 1118 OG SER B 55 −12.311 −27.509 2.717 1.00 78.36 O ATOM 1119 N GLY B 56 −11.001 −24.520 3.565 1.00 66.16 N ATOM 1120 CA GLY B 56 −11.144 −23.146 3.100 1.00 65.31 C ATOM 1121 C GLY B 56 −10.328 −22.147 3.935 1.00 67.72 C ATOM 1122 O GLY B 56 −10.112 −21.031 3.461 1.00 68.58 O ATOM 1123 N VAL B 57 −9.859 −22.529 5.153 1.00 61.01 N ATOM 1124 CA VAL B 57 −9.096 −21.625 6.021 1.00 59.84 C ATOM 1125 C VAL B 57 −7.686 −21.461 5.431 1.00 65.21 C ATOM 1126 O VAL B 57 −6.979 −22.458 5.339 1.00 65.74 O ATOM 1127 CB VAL B 57 −9.067 −22.123 7.490 1.00 61.18 C ATOM 1128 CG1 VAL B 57 −8.169 −21.243 8.361 1.00 61.69 C ATOM 1129 CG2 VAL B 57 −10.480 −22.183 8.063 1.00 57.87 C ATOM 1130 N PRO B 58 −7.278 −20.227 5.021 1.00 63.08 N ATOM 1131 CA PRO B 58 −5.952 −20.011 4.412 1.00 65.68 C ATOM 1132 C PRO B 58 −4.745 −20.491 5.234 1.00 70.54 C ATOM 1133 O PRO B 58 −4.815 −20.562 6.460 1.00 68.54 O ATOM 1134 CB PRO B 58 −5.914 −18.495 4.205 1.00 67.57 C ATOM 1135 CG PRO B 58 −7.318 −18.108 4.068 1.00 69.05 C ATOM 1136 CD PRO B 58 −8.033 −18.957 5.058 1.00 62.40 C ATOM 1137 N ALA B 59 −3.632 −20.796 4.535 1.00 70.63 N ATOM 1138 CA ALA B 59 −2.378 −21.291 5.132 1.00 73.06 C ATOM 1139 C ALA B 59 −1.738 −20.325 6.141 1.00 76.94 C ATOM 1140 O ALA B 59 −0.988 −20.775 7.012 1.00 77.91 O ATOM 1141 CB ALA B 59 −1.371 −21.620 4.035 1.00 77.06 C ATOM 1142 N ARG B 60 −2.010 −19.005 6.013 1.00 71.54 N ATOM 1143 CA ARG B 60 −1.493 −17.995 6.944 1.00 71.68 C ATOM 1144 C ARG B 60 −1.978 −18.201 8.384 1.00 73.05 C ATOM 1145 O ARG B 60 −1.354 −17.669 9.298 1.00 74.05 O ATOM 1146 CB ARG B 60 −1.822 −16.567 6.472 1.00 71.21 C ATOM 1147 CG ARG B 60 −3.305 −16.208 6.461 1.00 74.34 C ATOM 1148 CD ARG B 60 −3.505 −14.781 6.006 1.00 78.85 C ATOM 1149 NE ARG B 60 −4.898 −14.493 5.674 1.00 77.99 N ATOM 1150 CZ ARG B 60 −5.524 −14.853 4.555 1.00 87.93 C ATOM 1151 NH1 ARG B 60 −4.881 −15.533 3.608 1.00 76.36 N ATOM 1152 NH2 ARG B 60 −6.800 −14.533 4.370 1.00 67.82 N ATOM 1153 N PHE B 61 −3.088 −18.948 8.588 1.00 66.10 N ATOM 1154 CA PHE B 61 −3.597 −19.258 9.921 1.00 64.28 C ATOM 1155 C PHE B 61 −2.885 −20.476 10.488 1.00 69.61 C ATOM 1156 O PHE B 61 −2.547 −21.396 9.745 1.00 70.31 O ATOM 1157 CB PHE B 61 −5.106 −19.540 9.894 1.00 62.73 C ATOM 1158 CG PHE B 61 −5.943 −18.297 9.759 1.00 62.60 C ATOM 1159 CD1 PHE B 61 −6.368 −17.604 10.883 1.00 64.47 C ATOM 1160 CD2 PHE B 61 −6.301 −17.815 8.508 1.00 64.32 C ATOM 1161 CE1 PHE B 61 −7.151 −16.457 10.758 1.00 64.38 C ATOM 1162 CE2 PHE B 61 −7.079 −16.670 8.384 1.00 65.84 C ATOM 1163 CZ PHE B 61 −7.497 −15.994 9.509 1.00 63.15 C ATOM 1164 N SER B 62 −2.678 −20.487 11.806 1.00 66.32 N ATOM 1165 CA SER B 62 −2.061 −21.614 12.499 1.00 67.24 C ATOM 1166 C SER B 62 −2.434 −21.592 13.972 1.00 69.68 C ATOM 1167 O SER B 62 −2.704 −20.526 14.521 1.00 69.05 O ATOM 1168 CB SER B 62 −0.542 −21.602 12.332 1.00 74.31 C ATOM 1169 OG SER B 62 0.052 −20.447 12.904 1.00 85.34 O ATOM 1170 N GLY B 63 −2.459 −22.771 14.602 1.00 65.57 N ATOM 1171 CA GLY B 63 −2.791 −22.909 16.014 1.00 64.13 C ATOM 1172 C GLY B 63 −1.791 −23.831 16.702 1.00 70.06 C ATOM 1173 O GLY B 63 −1.291 −24.772 16.088 1.00 70.03 O ATOM 1174 N SER B 64 −1.483 −23.533 17.969 1.00 67.66 N ATOM 1175 CA SER B 64 −0.557 −24.305 18.792 1.00 69.46 C ATOM 1176 C SER B 64 −0.968 −24.184 20.254 1.00 72.46 C ATOM 1177 O SER B 64 −1.911 −23.456 20.567 1.00 70.44 O ATOM 1178 CB SER B 64 0.875 −23.801 18.600 1.00 77.56 C ATOM 1179 OG SER B 64 1.076 −22.504 19.141 1.00 86.08 O ATOM 1180 N GLY B 65 −0.266 −24.887 21.146 1.00 71.07 N ATOM 1181 CA GLY B 65 −0.564 −24.838 22.572 1.00 70.85 C ATOM 1182 C GLY B 65 −0.349 −26.161 23.270 1.00 75.44 C ATOM 1183 O GLY B 65 −0.241 −27.212 22.635 1.00 74.06 O ATOM 1184 N SER B 66 −0.318 −26.100 24.595 1.00 74.55 N ATOM 1185 CA SER B 66 −0.084 −27.265 25.435 1.00 76.47 C ATOM 1186 C SER B 66 −0.573 −26.987 26.858 1.00 81.99 C ATOM 1187 O SER B 66 −0.439 −25.863 27.346 1.00 83.12 O ATOM 1188 CB SER B 66 1.412 −27.586 25.451 1.00 83.75 C ATOM 1189 OG SER B 66 1.732 −28.667 26.311 1.00 94.88 O ATOM 1190 N GLY B 67 −1.130 −28.014 27.516 1.00 77.57 N ATOM 1191 CA GLY B 67 −1.587 −27.938 28.901 1.00 77.30 C ATOM 1192 C GLY B 67 −2.581 −26.821 29.210 1.00 78.60 C ATOM 1193 O GLY B 67 −3.778 −27.002 29.010 1.00 74.79 O ATOM 1194 N THR B 68 −2.084 −25.682 29.723 1.00 77.44 N ATOM 1195 CA THR B 68 −2.912 −24.563 30.180 1.00 76.28 C ATOM 1196 C THR B 68 −2.919 −23.336 29.241 1.00 79.65 C ATOM 1197 O THR B 68 −3.811 −22.505 29.399 1.00 77.30 O ATOM 1198 CB THR B 68 −2.471 −24.196 31.623 1.00 89.54 C ATOM 1199 OG1 THR B 68 −3.619 −23.956 32.434 1.00 89.46 O ATOM 1200 CG2 THR B 68 −1.510 −23.008 31.695 1.00 91.77 C ATOM 1201 N ASP B 69 −1.945 −23.192 28.312 1.00 77.83 N ATOM 1202 CA ASP B 69 −1.855 −22.026 27.418 1.00 77.02 C ATOM 1203 C ASP B 69 −1.973 −22.433 25.949 1.00 78.50 C ATOM 1204 O ASP B 69 −1.252 −23.329 25.511 1.00 80.10 O ATOM 1205 CB ASP B 69 −0.532 −21.282 27.653 1.00 82.57 C ATOM 1206 CG ASP B 69 −0.404 −20.722 29.054 1.00 96.01 C ATOM 1207 OD1 ASP B 69 −1.224 −19.854 29.423 1.00 95.53 O ATOM 1208 OD2 ASP B 69 0.510 −21.157 29.785 1.00 106.12 O ATOM 1209 N TYR B 70 −2.890 −21.780 25.197 1.00 70.97 N ATOM 1210 CA TYR B 70 −3.147 −22.053 23.777 1.00 68.56 C ATOM 1211 C TYR B 70 −3.176 −20.765 22.956 1.00 73.46 C ATOM 1212 O TYR B 70 −3.391 −19.688 23.517 1.00 73.56 O ATOM 1213 CB TYR B 70 −4.463 −22.830 23.619 1.00 65.52 C ATOM 1214 CG TYR B 70 −4.297 −24.297 23.937 1.00 65.72 C ATOM 1215 CD1 TYR B 70 −4.220 −24.739 25.252 1.00 67.72 C ATOM 1216 CD2 TYR B 70 −4.123 −25.232 22.926 1.00 65.72 C ATOM 1217 CE1 TYR B 70 −3.976 −26.073 25.551 1.00 67.27 C ATOM 1218 CE2 TYR B 70 −3.886 −26.573 23.214 1.00 66.49 C ATOM 1219 CZ TYR B 70 −3.828 −26.993 24.531 1.00 72.77 C ATOM 1220 OH TYR B 70 −3.617 −28.316 24.842 1.00 73.72 O ATOM 1221 N SER B 71 −2.924 −20.868 21.632 1.00 70.12 N ATOM 1222 CA SER B 71 −2.878 −19.692 20.758 1.00 70.18 C ATOM 1223 C SER B 71 −3.355 −19.946 19.329 1.00 71.24 C ATOM 1224 O SER B 71 −3.308 −21.073 18.841 1.00 69.77 O ATOM 1225 CB SER B 71 −1.452 −19.147 20.684 1.00 77.69 C ATOM 1226 OG SER B 71 −0.894 −18.886 21.961 1.00 90.01 O ATOM 1227 N LEU B 72 −3.774 −18.857 18.656 1.00 66.31 N ATOM 1228 CA LEU B 72 −4.166 −18.827 17.245 1.00 64.79 C ATOM 1229 C LEU B 72 −3.331 −17.692 16.640 1.00 70.72 C ATOM 1230 O LEU B 72 −3.393 −16.572 17.149 1.00 71.03 O ATOM 1231 CB LEU B 72 −5.676 −18.560 17.090 1.00 61.72 C ATOM 1232 CG LEU B 72 −6.245 −18.564 15.651 1.00 64.98 C ATOM 1233 CD1 LEU B 72 −7.662 −19.108 15.623 1.00 61.85 C ATOM 1234 CD2 LEU B 72 −6.256 −17.157 15.027 1.00 66.99 C ATOM 1235 N THR B 73 −2.517 −17.984 15.603 1.00 68.65 N ATOM 1236 CA THR B 73 −1.624 −16.999 14.978 1.00 70.53 C ATOM 1237 C THR B 73 −1.968 −16.754 13.501 1.00 73.74 C ATOM 1238 O THR B 73 −2.367 −17.680 12.800 1.00 72.13 O ATOM 1239 CB THR B 73 −0.162 −17.471 15.160 1.00 79.88 C ATOM 1240 OG1 THR B 73 0.157 −17.433 16.549 1.00 81.62 O ATOM 1241 CG2 THR B 73 0.857 −16.624 14.394 1.00 79.28 C ATOM 1242 N ILE B 74 −1.778 −15.505 13.030 1.00 71.50 N ATOM 1243 CA ILE B 74 −1.980 −15.113 11.628 1.00 71.40 C ATOM 1244 C ILE B 74 −0.664 −14.464 11.194 1.00 79.41 C ATOM 1245 O ILE B 74 −0.290 −13.440 11.761 1.00 79.98 O ATOM 1246 CB ILE B 74 −3.197 −14.168 11.438 1.00 71.75 C ATOM 1247 CG1 ILE B 74 −4.418 −14.696 12.228 1.00 68.88 C ATOM 1248 CG2 ILE B 74 −3.526 −14.020 9.941 1.00 71.80 C ATOM 1249 CD1 ILE B 74 −5.588 −13.837 12.206 1.00 73.61 C ATOM 1250 N SER B 75 0.071 −15.090 10.252 1.00 78.64 N ATOM 1251 CA SER B 75 1.388 −14.603 9.817 1.00 82.42 C ATOM 1252 C SER B 75 1.347 −13.211 9.178 1.00 87.14 C ATOM 1253 O SER B 75 2.162 −12.356 9.533 1.00 88.95 O ATOM 1254 CB SER B 75 2.051 −15.607 8.875 1.00 87.62 C ATOM 1255 OG SER B 75 1.248 −15.869 7.737 1.00 94.12 O ATOM 1256 N SER B 76 0.401 −12.988 8.252 1.00 82.09 N ATOM 1257 CA SER B 76 0.217 −11.700 7.567 1.00 82.20 C ATOM 1258 C SER B 76 −1.282 −11.441 7.393 1.00 82.22 C ATOM 1259 O SER B 76 −1.908 −12.063 6.537 1.00 80.72 O ATOM 1260 CB SER B 76 0.920 −11.708 6.211 1.00 88.43 C ATOM 1261 OG SER B 76 0.681 −10.512 5.486 1.00 96.36 O ATOM 1262 N LEU B 77 −1.850 −10.528 8.209 1.00 76.84 N ATOM 1263 CA LEU B 77 −3.283 −10.196 8.203 1.00 72.92 C ATOM 1264 C LEU B 77 −3.815 −9.691 6.857 1.00 77.19 C ATOM 1265 O LEU B 77 −3.216 −8.799 6.259 1.00 78.61 O ATOM 1266 CB LEU B 77 −3.589 −9.118 9.261 1.00 72.06 C ATOM 1267 CG LEU B 77 −3.456 −9.526 10.722 1.00 76.55 C ATOM 1268 CD1 LEU B 77 −3.193 −8.312 11.600 1.00 77.54 C ATOM 1269 CD2 LEU B 77 −4.691 −10.251 11.198 1.00 75.30 C ATOM 1270 N GLU B 78 −4.972 −10.225 6.424 1.00 72.28 N ATOM 1271 CA GLU B 78 −5.711 −9.801 5.229 1.00 71.21 C ATOM 1272 C GLU B 78 −7.040 −9.187 5.736 1.00 70.61 C ATOM 1273 O GLU B 78 −7.466 −9.537 6.843 1.00 68.30 O ATOM 1274 CB GLU B 78 −6.026 −10.996 4.312 1.00 72.49 C ATOM 1275 CG GLU B 78 −4.812 −11.635 3.656 1.00 87.48 C ATOM 1276 CD GLU B 78 −4.022 −10.763 2.703 1.00 112.73 C ATOM 1277 OE1 GLU B 78 −4.620 −9.856 2.080 1.00 110.91 O ATOM 1278 OE2 GLU B 78 −2.805 −11.016 2.551 1.00 112.15 O ATOM 1279 N PRO B 79 −7.706 −8.302 4.944 1.00 65.02 N ATOM 1280 CA PRO B 79 −8.970 −7.679 5.373 1.00 61.28 C ATOM 1281 C PRO B 79 −10.086 −8.645 5.834 1.00 60.72 C ATOM 1282 O PRO B 79 −10.835 −8.311 6.751 1.00 58.19 O ATOM 1283 CB PRO B 79 −9.396 −6.879 4.140 1.00 62.94 C ATOM 1284 CG PRO B 79 −8.135 −6.506 3.486 1.00 70.44 C ATOM 1285 CD PRO B 79 −7.172 −7.637 3.736 1.00 68.02 C ATOM 1286 N GLU B 80 −10.194 −9.824 5.203 1.00 56.30 N ATOM 1287 CA GLU B 80 −11.226 −10.813 5.534 1.00 53.71 C ATOM 1288 C GLU B 80 −11.016 −11.525 6.900 1.00 56.40 C ATOM 1289 O GLU B 80 −11.966 −12.110 7.424 1.00 53.35 O ATOM 1290 CB GLU B 80 −11.337 −11.857 4.403 1.00 55.59 C ATOM 1291 CG GLU B 80 −10.193 −12.863 4.378 1.00 68.69 C ATOM 1292 CD GLU B 80 −9.827 −13.412 3.013 1.00 97.64 C ATOM 1293 OE1 GLU B 80 −8.829 −12.932 2.427 1.00 100.61 O ATOM 1294 OE2 GLU B 80 −10.516 −14.347 2.544 1.00 91.35 O ATOM 1295 N ASP B 81 −9.797 −11.485 7.464 1.00 54.10 N ATOM 1296 CA ASP B 81 −9.474 −12.158 8.733 1.00 53.36 C ATOM 1297 C ASP B 81 −10.008 −11.455 9.967 1.00 56.43 C ATOM 1298 O ASP B 81 −10.130 −12.090 11.016 1.00 55.62 O ATOM 1299 CB ASP B 81 −7.949 −12.273 8.904 1.00 57.40 C ATOM 1300 CG ASP B 81 −7.242 −12.953 7.763 1.00 64.20 C ATOM 1301 OD1 ASP B 81 −7.891 −13.751 7.049 1.00 63.36 O ATOM 1302 OD2 ASP B 81 −6.048 −12.665 7.554 1.00 71.18 O ATOM 1303 N PHE B 82 −10.268 −10.151 9.878 1.00 52.95 N ATOM 1304 CA PHE B 82 −10.706 −9.365 11.027 1.00 51.87 C ATOM 1305 C PHE B 82 −12.131 −9.787 11.419 1.00 52.04 C ATOM 1306 O PHE B 82 −13.036 −9.733 10.591 1.00 50.63 O ATOM 1307 CB PHE B 82 −10.553 −7.861 10.735 1.00 54.14 C ATOM 1308 CG PHE B 82 −9.102 −7.462 10.536 1.00 58.34 C ATOM 1309 CD1 PHE B 82 −8.307 −7.110 11.615 1.00 62.40 C ATOM 1310 CD2 PHE B 82 −8.537 −7.435 9.269 1.00 61.95 C ATOM 1311 CE1 PHE B 82 −6.971 −6.754 11.435 1.00 65.79 C ATOM 1312 CE2 PHE B 82 −7.199 −7.073 9.089 1.00 67.27 C ATOM 1313 CZ PHE B 82 −6.428 −6.725 10.173 1.00 66.48 C ATOM 1314 N ALA B 83 −12.285 −10.316 12.648 1.00 46.96 N ATOM 1315 CA ALA B 83 −13.532 −10.898 13.174 1.00 44.03 C ATOM 1316 C ALA B 83 −13.345 −11.212 14.673 1.00 47.01 C ATOM 1317 O ALA B 83 −12.292 −10.889 15.225 1.00 48.83 O ATOM 1318 CB ALA B 83 −13.837 −12.202 12.423 1.00 43.57 C ATOM 1319 N VAL B 84 −14.340 −11.848 15.324 1.00 41.13 N ATOM 1320 CA VAL B 84 −14.216 −12.290 16.722 1.00 40.65 C ATOM 1321 C VAL B 84 −13.793 −13.754 16.685 1.00 44.54 C ATOM 1322 O VAL B 84 −14.269 −14.500 15.831 1.00 43.86 O ATOM 1323 CB VAL B 84 −15.510 −12.086 17.558 1.00 41.88 C ATOM 1324 CG1 VAL B 84 −15.368 −12.686 18.961 1.00 41.89 C ATOM 1325 CG2 VAL B 84 −15.860 −10.604 17.656 1.00 40.84 C ATOM 1326 N TYR B 85 −12.893 −14.159 17.588 1.00 42.41 N ATOM 1327 CA TYR B 85 −12.420 −15.541 17.671 1.00 42.97 C ATOM 1328 C TYR B 85 −12.637 −16.085 19.063 1.00 48.03 C ATOM 1329 O TYR B 85 −12.177 −15.468 20.020 1.00 49.51 O ATOM 1330 CB TYR B 85 −10.939 −15.628 17.307 1.00 45.67 C ATOM 1331 CG TYR B 85 −10.707 −15.379 15.838 1.00 45.85 C ATOM 1332 CD1 TYR B 85 −10.551 −14.087 15.348 1.00 47.32 C ATOM 1333 CD2 TYR B 85 −10.708 −16.426 14.926 1.00 45.53 C ATOM 1334 CE1 TYR B 85 −10.359 −13.848 13.993 1.00 47.60 C ATOM 1335 CE2 TYR B 85 −10.518 −16.201 13.567 1.00 46.72 C ATOM 1336 CZ TYR B 85 −10.336 −14.909 13.102 1.00 53.44 C ATOM 1337 OH TYR B 85 −10.188 −14.702 11.751 1.00 51.93 O ATOM 1338 N TYR B 86 −13.323 −17.236 19.182 1.00 43.51 N ATOM 1339 CA TYR B 86 −13.582 −17.888 20.470 1.00 43.19 C ATOM 1340 C TYR B 86 −12.844 −19.192 20.544 1.00 47.49 C ATOM 1341 O TYR B 86 −12.954 −19.977 19.609 1.00 45.45 O ATOM 1342 CB TYR B 86 −15.072 −18.211 20.631 1.00 41.61 C ATOM 1343 CG TYR B 86 −15.977 −17.004 20.629 1.00 40.75 C ATOM 1344 CD1 TYR B 86 −16.332 −16.376 21.813 1.00 42.52 C ATOM 1345 CD2 TYR B 86 −16.554 −16.546 19.450 1.00 39.56 C ATOM 1346 CE1 TYR B 86 −17.226 −15.309 21.827 1.00 43.46 C ATOM 1347 CE2 TYR B 86 −17.443 −15.475 19.448 1.00 39.13 C ATOM 1348 CZ TYR B 86 −17.763 −14.845 20.638 1.00 46.08 C ATOM 1349 OH TYR B 86 −18.644 −13.793 20.669 1.00 45.53 O ATOM 1350 N CYS B 87 −12.158 −19.466 21.668 1.00 47.69 N ATOM 1351 CA CYS B 87 −11.518 −20.760 21.891 1.00 48.62 C ATOM 1352 C CYS B 87 −12.560 −21.608 22.630 1.00 49.15 C ATOM 1353 O CYS B 87 −13.528 −21.055 23.154 1.00 46.52 O ATOM 1354 CB CYS B 87 −10.190 −20.652 22.652 1.00 52.40 C ATOM 1355 SG CYS B 87 −10.286 −19.868 24.288 1.00 57.94 S ATOM 1356 N GLN B 88 −12.424 −22.936 22.593 1.00 45.22 N ATOM 1357 CA GLN B 88 −13.372 −23.849 23.241 1.00 42.45 C ATOM 1358 C GLN B 88 −12.691 −25.152 23.625 1.00 46.38 C ATOM 1359 O GLN B 88 −11.897 −25.674 22.843 1.00 47.02 O ATOM 1360 CB GLN B 88 −14.529 −24.155 22.280 1.00 40.68 C ATOM 1361 CG GLN B 88 −15.647 −25.055 22.846 1.00 42.62 C ATOM 1362 CD GLN B 88 −15.772 −26.410 22.162 1.00 51.06 C ATOM 1363 OE1 GLN B 88 −15.816 −26.515 20.933 1.00 42.02 O ATOM 1364 NE2 GLN B 88 −15.948 −27.479 22.927 1.00 41.44 N ATOM 1365 N GLN B 89 −13.041 −25.708 24.797 1.00 42.41 N ATOM 1366 CA GLN B 89 −12.504 −26.993 25.248 1.00 42.78 C ATOM 1367 C GLN B 89 −13.621 −28.045 25.323 1.00 45.27 C ATOM 1368 O GLN B 89 −14.790 −27.709 25.533 1.00 42.06 O ATOM 1369 CB GLN B 89 −11.731 −26.856 26.583 1.00 45.86 C ATOM 1370 CG GLN B 89 −12.566 −26.473 27.816 1.00 48.91 C ATOM 1371 CD GLN B 89 −13.360 −27.612 28.428 1.00 58.10 C ATOM 1372 OE1 GLN B 89 −12.980 −28.785 28.353 1.00 50.12 O ATOM 1373 NE2 GLN B 89 −14.473 −27.295 29.079 1.00 46.79 N ATOM 1374 N TRP B 90 −13.240 −29.317 25.149 1.00 43.48 N ATOM 1375 CA TRP B 90 −14.143 −30.472 25.206 1.00 42.34 C ATOM 1376 C TRP B 90 −13.533 −31.605 26.079 1.00 48.13 C ATOM 1377 O TRP B 90 −13.926 −32.759 25.939 1.00 47.04 O ATOM 1378 CB TRP B 90 −14.479 −30.972 23.778 1.00 39.82 C ATOM 1379 CG TRP B 90 −13.291 −31.452 22.996 1.00 42.22 C ATOM 1380 CD1 TRP B 90 −12.282 −30.690 22.486 1.00 46.21 C ATOM 1381 CD2 TRP B 90 −12.984 −32.808 22.650 1.00 42.49 C ATOM 1382 NE1 TRP B 90 −11.355 −31.487 21.862 1.00 46.65 N ATOM 1383 CE2 TRP B 90 −11.761 −32.794 21.946 1.00 47.73 C ATOM 1384 CE3 TRP B 90 −13.644 −34.033 22.834 1.00 43.26 C ATOM 1385 CZ2 TRP B 90 −11.184 −33.957 21.427 1.00 47.77 C ATOM 1386 CZ3 TRP B 90 −13.064 −35.187 22.333 1.00 45.41 C ATOM 1387 CH2 TRP B 90 −11.848 −35.143 21.641 1.00 47.43 C ATOM 1388 N ASN B 91 −12.614 −31.261 27.004 1.00 47.97 N ATOM 1389 CA ASN B 91 −11.971 −32.226 27.903 1.00 50.26 C ATOM 1390 C ASN B 91 −12.892 −32.662 29.018 1.00 54.80 C ATOM 1391 O ASN B 91 −12.780 −33.797 29.489 1.00 53.62 O ATOM 1392 CB ASN B 91 −10.692 −31.638 28.519 1.00 54.00 C ATOM 1393 CG ASN B 91 −9.549 −31.598 27.549 1.00 78.58 C ATOM 1394 OD1 ASN B 91 −9.320 −30.601 26.869 1.00 75.71 O ATOM 1395 ND2 ASN B 91 −8.840 −32.704 27.422 1.00 73.65 N ATOM 1396 O SER B 92 −15.973 −30.214 29.820 1.00 53.47 O ATOM 1397 N SER B 92 −13.750 −31.747 29.497 1.00 52.61 N ATOM 1398 CA SER B 92 −14.671 −32.051 30.590 1.00 52.75 C ATOM 1399 C SER B 92 −15.962 −31.270 30.446 1.00 53.91 C ATOM 1400 CB SER B 92 −14.018 −31.722 31.930 1.00 58.69 C ATOM 1401 OG SER B 92 −13.721 −30.337 32.038 1.00 67.43 O ATOM 1402 N TYR B 93 −17.048 −31.801 31.019 1.00 49.07 N ATOM 1403 CA TYR B 93 −18.350 −31.144 31.006 1.00 47.17 C ATOM 1404 C TYR B 93 −18.430 −30.177 32.203 1.00 50.89 C ATOM 1405 O TYR B 93 −17.932 −30.514 33.281 1.00 51.68 O ATOM 1406 CB TYR B 93 −19.496 −32.162 31.091 1.00 47.60 C ATOM 1407 CG TYR B 93 −19.624 −33.024 29.857 1.00 48.53 C ATOM 1408 CD1 TYR B 93 −19.987 −32.475 28.633 1.00 48.47 C ATOM 1409 CD2 TYR B 93 −19.428 −34.399 29.922 1.00 50.28 C ATOM 1410 CE1 TYR B 93 −20.128 −33.267 27.495 1.00 48.36 C ATOM 1411 CE2 TYR B 93 −19.573 −35.205 28.793 1.00 50.27 C ATOM 1412 CZ TYR B 93 −19.929 −34.636 27.580 1.00 52.39 C ATOM 1413 OH TYR B 93 −20.053 −35.409 26.449 1.00 47.02 O ATOM 1414 N PRO B 94 −19.053 −28.994 32.034 1.00 45.75 N ATOM 1415 CA PRO B 94 −19.670 −28.475 30.807 1.00 43.75 C ATOM 1416 C PRO B 94 −18.618 −27.971 29.813 1.00 46.73 C ATOM 1417 O PRO B 94 −17.566 −27.486 30.232 1.00 47.63 O ATOM 1418 CB PRO B 94 −20.561 −27.334 31.324 1.00 45.47 C ATOM 1419 CG PRO B 94 −19.843 −26.838 32.548 1.00 51.32 C ATOM 1420 CD PRO B 94 −19.241 −28.071 33.172 1.00 47.46 C ATOM 1421 N PHE B 95 −18.877 −28.116 28.496 1.00 40.21 N ATOM 1422 CA PHE B 95 −17.969 −27.570 27.485 1.00 39.48 C ATOM 1423 C PHE B 95 −18.007 −26.068 27.663 1.00 43.23 C ATOM 1424 O PHE B 95 −19.069 −25.524 27.959 1.00 41.42 O ATOM 1425 CB PHE B 95 −18.397 −27.951 26.059 1.00 39.28 C ATOM 1426 CG PHE B 95 −18.239 −29.399 25.644 1.00 40.62 C ATOM 1427 CD1 PHE B 95 −17.686 −30.340 26.511 1.00 44.44 C ATOM 1428 CD2 PHE B 95 −18.659 −29.827 24.394 1.00 41.19 C ATOM 1429 CE1 PHE B 95 −17.507 −31.660 26.107 1.00 45.39 C ATOM 1430 CE2 PHE B 95 −18.494 −31.153 24.001 1.00 43.39 C ATOM 1431 CZ PHE B 95 −17.911 −32.057 24.857 1.00 42.95 C ATOM 1432 N THR B 96 −16.855 −25.407 27.572 1.00 41.95 N ATOM 1433 CA THR B 96 −16.784 −23.968 27.795 1.00 42.74 C ATOM 1434 C THR B 96 −16.094 −23.273 26.668 1.00 47.87 C ATOM 1435 O THR B 96 −15.230 −23.845 26.011 1.00 48.17 O ATOM 1436 CB THR B 96 −16.095 −23.646 29.123 1.00 48.68 C ATOM 1437 OG1 THR B 96 −14.905 −24.428 29.244 1.00 48.40 O ATOM 1438 CG2 THR B 96 −16.998 −23.889 30.307 1.00 47.75 C ATOM 1439 N PHE B 97 −16.462 −22.013 26.477 1.00 43.76 N ATOM 1440 CA PHE B 97 −15.899 −21.142 25.466 1.00 43.33 C ATOM 1441 C PHE B 97 −15.188 −20.007 26.161 1.00 50.88 C ATOM 1442 O PHE B 97 −15.564 −19.625 27.271 1.00 51.63 O ATOM 1443 CB PHE B 97 −17.023 −20.552 24.605 1.00 42.06 C ATOM 1444 CG PHE B 97 −17.737 −21.534 23.708 1.00 40.37 C ATOM 1445 CD1 PHE B 97 −17.321 −21.728 22.397 1.00 40.99 C ATOM 1446 CD2 PHE B 97 −18.901 −22.164 24.129 1.00 39.39 C ATOM 1447 CE1 PHE B 97 −18.026 −22.572 21.542 1.00 39.83 C ATOM 1448 CE2 PHE B 97 −19.613 −22.996 23.267 1.00 39.99 C ATOM 1449 CZ PHE B 97 −19.163 −23.206 21.985 1.00 37.63 C ATOM 1450 N GLY B 98 −14.202 −19.425 25.494 1.00 49.49 N ATOM 1451 CA GLY B 98 −13.544 −18.227 26.001 1.00 51.19 C ATOM 1452 C GLY B 98 −14.500 −17.039 25.742 1.00 52.41 C ATOM 1453 O GLY B 98 −15.479 −17.187 25.010 1.00 47.63 O ATOM 1454 N GLN B 99 −14.237 −15.881 26.362 1.00 50.95 N ATOM 1455 CA GLN B 99 −15.097 −14.696 26.206 1.00 48.95 C ATOM 1456 C GLN B 99 −15.001 −14.060 24.797 1.00 51.79 C ATOM 1457 O GLN B 99 −15.858 −13.248 24.437 1.00 50.22 O ATOM 1458 CB GLN B 99 −14.835 −13.670 27.332 1.00 51.64 C ATOM 1459 CG GLN B 99 −13.528 −12.869 27.232 1.00 68.39 C ATOM 1460 CD GLN B 99 −12.368 −13.491 27.964 1.00 85.35 C ATOM 1461 OE1 GLN B 99 −12.224 −14.721 28.017 1.00 76.51 O ATOM 1462 NE2 GLN B 99 −11.464 −12.665 28.473 1.00 81.45 N ATOM 1463 N GLY B 100 −13.963 −14.416 24.017 1.00 48.68 N ATOM 1464 CA GLY B 100 −13.786 −13.935 22.652 1.00 47.98 C ATOM 1465 C GLY B 100 −12.769 −12.800 22.563 1.00 54.37 C ATOM 1466 O GLY B 100 −12.646 −11.999 23.490 1.00 55.57 O ATOM 1467 N THR B 101 −12.051 −12.737 21.434 1.00 51.18 N ATOM 1468 CA THR B 101 −11.072 −11.694 21.142 1.00 52.85 C ATOM 1469 C THR B 101 −11.459 −11.061 19.815 1.00 55.49 C ATOM 1470 O THR B 101 −11.479 −11.759 18.800 1.00 54.25 O ATOM 1471 CB THR B 101 −9.657 −12.288 21.088 1.00 59.98 C ATOM 1472 OG1 THR B 101 −9.238 −12.578 22.418 1.00 58.56 O ATOM 1473 CG2 THR B 101 −8.639 −11.352 20.423 1.00 60.05 C ATOM 1474 N LYS B 102 −11.749 −9.752 19.808 1.00 51.10 N ATOM 1475 CA LYS B 102 −12.081 −9.066 18.561 1.00 49.56 C ATOM 1476 C LYS B 102 −10.785 −8.679 17.860 1.00 55.44 C ATOM 1477 O LYS B 102 −10.052 −7.823 18.352 1.00 56.75 O ATOM 1478 CB LYS B 102 −12.957 −7.818 18.794 1.00 50.04 C ATOM 1479 CG LYS B 102 −13.420 −7.168 17.489 1.00 50.49 C ATOM 1480 CD LYS B 102 −14.335 −5.972 17.713 1.00 55.49 C ATOM 1481 CE LYS B 102 −14.869 −5.432 16.407 1.00 63.54 C ATOM 1482 NZ LYS B 102 −15.701 −4.212 16.594 1.00 70.94 N ATOM 1483 N LEU B 103 −10.503 −9.321 16.718 1.00 51.50 N ATOM 1484 CA LEU B 103 −9.348 −8.997 15.892 1.00 52.92 C ATOM 1485 C LEU B 103 −9.859 −7.856 15.011 1.00 56.83 C ATOM 1486 O LEU B 103 −10.659 −8.077 14.099 1.00 55.12 O ATOM 1487 CB LEU B 103 −8.896 −10.225 15.088 1.00 53.04 C ATOM 1488 CG LEU B 103 −7.818 −10.012 14.047 1.00 59.35 C ATOM 1489 CD1 LEU B 103 −6.578 −9.370 14.644 1.00 62.10 C ATOM 1490 CD2 LEU B 103 −7.470 −11.318 13.391 1.00 60.99 C ATOM 1491 N GLU B 104 −9.461 −6.624 15.368 1.00 54.35 N ATOM 1492 CA GLU B 104 −9.941 −5.362 14.801 1.00 52.67 C ATOM 1493 C GLU B 104 −8.902 −4.667 13.895 1.00 59.92 C ATOM 1494 O GLU B 104 −7.704 −4.819 14.125 1.00 62.89 O ATOM 1495 CB GLU B 104 −10.306 −4.472 16.000 1.00 53.24 C ATOM 1496 CG GLU B 104 −11.095 −3.228 15.665 1.00 60.41 C ATOM 1497 CD GLU B 104 −10.419 −1.909 15.975 1.00 77.51 C ATOM 1498 OE1 GLU B 104 −9.176 −1.816 15.854 1.00 73.51 O ATOM 1499 OE2 GLU B 104 −11.143 −0.959 16.346 1.00 65.92 O ATOM 1500 N ILE B 105 −9.352 −3.948 12.844 1.00 55.66 N ATOM 1501 CA ILE B 105 −8.434 −3.263 11.918 1.00 58.22 C ATOM 1502 C ILE B 105 −7.838 −2.002 12.552 1.00 63.24 C ATOM 1503 O ILE B 105 −8.589 −1.115 12.955 1.00 61.14 O ATOM 1504 CB ILE B 105 −9.109 −2.922 10.556 1.00 60.89 C ATOM 1505 CG1 ILE B 105 −9.492 −4.210 9.816 1.00 60.92 C ATOM 1506 CG2 ILE B 105 −8.161 −2.075 9.682 1.00 64.50 C ATOM 1507 CD1 ILE B 105 −10.268 −4.039 8.503 1.00 70.71 C ATOM 1508 N LYS B 106 −6.495 −1.884 12.551 1.00 63.25 N ATOM 1509 CA LYS B 106 −5.810 −0.686 13.041 1.00 65.26 C ATOM 1510 C LYS B 106 −5.658 0.221 11.842 1.00 70.42 C ATOM 1511 O LYS B 106 −5.169 −0.195 10.787 1.00 71.21 O ATOM 1512 CB LYS B 106 −4.453 −0.972 13.683 1.00 70.36 C ATOM 1513 CG LYS B 106 −3.914 0.227 14.467 1.00 85.15 C ATOM 1514 CD LYS B 106 −2.697 −0.113 15.335 1.00 99.27 C ATOM 1515 CE LYS B 106 −2.774 0.524 16.703 1.00 111.93 C ATOM 1516 NZ LYS B 106 −1.600 0.181 17.549 1.00 126.28 N ATOM 1517 N ARG B 107 −6.111 1.453 12.007 1.00 66.11 N ATOM 1518 CA ARG B 107 −6.185 2.465 10.973 1.00 65.54 C ATOM 1519 C ARG B 107 −5.486 3.722 11.451 1.00 70.00 C ATOM 1520 O ARG B 107 −5.151 3.820 12.635 1.00 70.45 O ATOM 1521 CB ARG B 107 −7.676 2.770 10.767 1.00 63.82 C ATOM 1522 CG ARG B 107 −8.080 2.936 9.334 1.00 71.44 C ATOM 1523 CD ARG B 107 −9.246 3.880 9.209 1.00 69.88 C ATOM 1524 NE ARG B 107 −8.786 5.273 9.205 1.00 77.15 N ATOM 1525 CZ ARG B 107 −9.155 6.218 8.338 1.00 91.04 C ATOM 1526 NH1 ARG B 107 −10.041 5.954 7.382 1.00 76.11 N ATOM 1527 NH2 ARG B 107 −8.647 7.441 8.431 1.00 81.81 N ATOM 1528 N THR B 108 −5.310 4.706 10.559 1.00 66.82 N ATOM 1529 CA THR B 108 −4.753 5.990 10.971 1.00 69.09 C ATOM 1530 C THR B 108 −5.872 6.696 11.738 1.00 71.48 C ATOM 1531 O THR B 108 −7.053 6.506 11.419 1.00 67.72 O ATOM 1532 CB THR B 108 −4.261 6.817 9.772 1.00 79.28 C ATOM 1533 OG1 THR B 108 −5.305 6.925 8.800 1.00 76.46 O ATOM 1534 CG2 THR B 108 −3.018 6.225 9.135 1.00 81.09 C ATOM 1535 N VAL B 109 −5.512 7.458 12.777 1.00 69.90 N ATOM 1536 CA VAL B 109 −6.497 8.166 13.592 1.00 67.67 C ATOM 1537 C VAL B 109 −7.332 9.092 12.703 1.00 70.41 C ATOM 1538 O VAL B 109 −6.775 9.815 11.872 1.00 72.05 O ATOM 1539 CB VAL B 109 −5.821 8.935 14.758 1.00 73.33 C ATOM 1540 CG1 VAL B 109 −6.773 9.952 15.396 1.00 71.74 C ATOM 1541 CG2 VAL B 109 −5.293 7.959 15.806 1.00 73.97 C ATOM 1542 N ALA B 110 −8.662 9.040 12.867 1.00 62.90 N ATOM 1543 CA ALA B 110 −9.602 9.877 12.127 1.00 61.06 C ATOM 1544 C ALA B 110 −10.496 10.573 13.134 1.00 64.18 C ATOM 1545 O ALA B 110 −11.096 9.905 13.968 1.00 63.18 O ATOM 1546 CB ALA B 110 −10.440 9.018 11.196 1.00 59.63 C ATOM 1547 N ALA B 111 −10.567 11.904 13.086 1.00 62.07 N ATOM 1548 CA ALA B 111 −11.396 12.672 14.016 1.00 61.29 C ATOM 1549 C ALA B 111 −12.875 12.567 13.619 1.00 61.25 C ATOM 1550 O ALA B 111 −13.164 12.492 12.424 1.00 59.38 O ATOM 1551 CB ALA B 111 −10.972 14.135 14.006 1.00 64.06 C ATOM 1552 N PRO B 112 −13.809 12.588 14.595 1.00 57.11 N ATOM 1553 CA PRO B 112 −15.230 12.525 14.248 1.00 54.57 C ATOM 1554 C PRO B 112 −15.769 13.860 13.745 1.00 57.94 C ATOM 1555 O PRO B 112 −15.499 14.899 14.349 1.00 57.84 O ATOM 1556 CB PRO B 112 −15.907 12.145 15.577 1.00 55.27 C ATOM 1557 CG PRO B 112 −14.998 12.664 16.619 1.00 61.79 C ATOM 1558 CD PRO B 112 −13.617 12.460 16.055 1.00 59.47 C ATOM 1559 N SER B 113 −16.571 13.819 12.672 1.00 52.50 N ATOM 1560 CA SER B 113 −17.291 14.996 12.197 1.00 51.51 C ATOM 1561 C SER B 113 −18.548 14.963 13.071 1.00 51.97 C ATOM 1562 O SER B 113 −19.288 13.970 13.036 1.00 49.85 O ATOM 1563 CB SER B 113 −17.653 14.872 10.720 1.00 53.97 C ATOM 1564 OG SER B 113 −16.477 14.810 9.930 1.00 66.05 O ATOM 1565 N VAL B 114 −18.716 15.965 13.939 1.00 46.85 N ATOM 1566 CA VAL B 114 −19.819 16.000 14.894 1.00 45.32 C ATOM 1567 C VAL B 114 −20.995 16.802 14.324 1.00 48.58 C ATOM 1568 O VAL B 114 −20.784 17.830 13.683 1.00 48.53 O ATOM 1569 CB VAL B 114 −19.346 16.530 16.277 1.00 50.19 C ATOM 1570 CG1 VAL B 114 −20.440 16.375 17.338 1.00 48.89 C ATOM 1571 CG2 VAL B 114 −18.077 15.805 16.728 1.00 51.02 C ATOM 1572 N PHE B 115 −22.231 16.308 14.534 1.00 44.22 N ATOM 1573 CA PHE B 115 −23.459 16.971 14.082 1.00 42.95 C ATOM 1574 C PHE B 115 −24.507 16.871 15.190 1.00 46.54 C ATOM 1575 O PHE B 115 −24.619 15.819 15.812 1.00 46.20 O ATOM 1576 CB PHE B 115 −24.014 16.310 12.798 1.00 43.15 C ATOM 1577 CG PHE B 115 −23.038 16.204 11.651 1.00 44.57 C ATOM 1578 CD1 PHE B 115 −22.203 15.101 11.526 1.00 46.42 C ATOM 1579 CD2 PHE B 115 −22.924 17.228 10.718 1.00 47.28 C ATOM 1580 CE1 PHE B 115 −21.272 15.022 10.490 1.00 47.73 C ATOM 1581 CE2 PHE B 115 −21.996 17.145 9.676 1.00 50.26 C ATOM 1582 CZ PHE B 115 −21.177 16.043 9.569 1.00 48.05 C ATOM 1583 N ILE B 116 −25.284 17.940 15.425 1.00 42.16 N ATOM 1584 CA ILE B 116 −26.345 17.930 16.435 1.00 41.52 C ATOM 1585 C ILE B 116 −27.683 18.171 15.742 1.00 44.82 C ATOM 1586 O ILE B 116 −27.770 19.015 14.854 1.00 45.21 O ATOM 1587 CB ILE B 116 −26.083 18.901 17.620 1.00 45.51 C ATOM 1588 CG1 ILE B 116 −27.075 18.616 18.773 1.00 44.96 C ATOM 1589 CG2 ILE B 116 −26.120 20.383 17.175 1.00 47.17 C ATOM 1590 CD1 ILE B 116 −26.788 19.383 20.097 1.00 50.62 C ATOM 1591 N PHE B 117 −28.719 17.415 16.130 1.00 40.34 N ATOM 1592 CA PHE B 117 −30.047 17.523 15.528 1.00 38.63 C ATOM 1593 C PHE B 117 −31.083 17.841 16.607 1.00 45.82 C ATOM 1594 O PHE B 117 −31.190 17.089 17.580 1.00 45.97 O ATOM 1595 CB PHE B 117 −30.411 16.208 14.822 1.00 37.51 C ATOM 1596 CG PHE B 117 −29.438 15.813 13.741 1.00 37.97 C ATOM 1597 CD1 PHE B 117 −29.533 16.356 12.467 1.00 39.10 C ATOM 1598 CD2 PHE B 117 −28.463 14.852 13.978 1.00 39.97 C ATOM 1599 CE1 PHE B 117 −28.676 15.942 11.445 1.00 40.25 C ATOM 1600 CE2 PHE B 117 −27.605 14.436 12.954 1.00 42.48 C ATOM 1601 CZ PHE B 117 −27.710 14.993 11.696 1.00 40.72 C ATOM 1602 N PRO B 118 −31.841 18.946 16.455 1.00 45.09 N ATOM 1603 CA PRO B 118 −32.879 19.259 17.437 1.00 45.31 C ATOM 1604 C PRO B 118 −34.092 18.334 17.227 1.00 49.18 C ATOM 1605 O PRO B 118 −34.194 17.684 16.182 1.00 46.92 O ATOM 1606 CB PRO B 118 −33.210 20.730 17.150 1.00 47.56 C ATOM 1607 CG PRO B 118 −32.884 20.932 15.748 1.00 51.11 C ATOM 1608 CD PRO B 118 −31.803 19.959 15.378 1.00 46.29 C ATOM 1609 N PRO B 119 −35.008 18.255 18.209 1.00 48.02 N ATOM 1610 CA PRO B 119 −36.187 17.422 17.994 1.00 47.42 C ATOM 1611 C PRO B 119 −37.046 18.010 16.858 1.00 50.41 C ATOM 1612 O PRO B 119 −37.065 19.219 16.645 1.00 51.03 O ATOM 1613 CB PRO B 119 −36.912 17.449 19.350 1.00 50.01 C ATOM 1614 CG PRO B 119 −36.196 18.448 20.190 1.00 55.19 C ATOM 1615 CD PRO B 119 −35.195 19.151 19.367 1.00 50.65 C ATOM 1616 N SER B 120 −37.724 17.149 16.127 1.00 46.48 N ATOM 1617 CA SER B 120 −38.611 17.562 15.045 1.00 45.63 C ATOM 1618 C SER B 120 −39.898 18.135 15.638 1.00 50.81 C ATOM 1619 O SER B 120 −40.253 17.812 16.769 1.00 49.99 O ATOM 1620 CB SER B 120 −38.947 16.366 14.161 1.00 45.74 C ATOM 1621 OG SER B 120 −39.828 15.466 14.817 1.00 48.70 O ATOM 1622 N ASP B 121 −40.604 18.967 14.868 1.00 49.41 N ATOM 1623 CA ASP B 121 −41.889 19.536 15.296 1.00 50.62 C ATOM 1624 C ASP B 121 −42.951 18.443 15.395 1.00 53.32 C ATOM 1625 O ASP B 121 −43.837 18.535 16.245 1.00 54.69 O ATOM 1626 CB ASP B 121 −42.348 20.651 14.339 1.00 53.45 C ATOM 1627 CG ASP B 121 −41.474 21.893 14.384 1.00 66.85 C ATOM 1628 OD1 ASP B 121 −40.793 22.109 15.419 1.00 66.32 O ATOM 1629 OD2 ASP B 121 −41.474 22.656 13.391 1.00 75.46 O ATOM 1630 N AGLU B 122 −42.837 17.400 14.554 0.50 48.70 N ATOM 1631 CA AGLU B 122 −43.751 16.256 14.541 0.50 48.23 C ATOM 1632 C AGLU B 122 −43.694 15.516 15.884 0.50 50.26 C ATOM 1633 O AGLU B 122 −44.740 15.186 16.440 0.50 50.69 O ATOM 1634 CB AGLU B 122 −43.392 15.278 13.399 0.50 48.42 C ATOM 1635 CG AGLU B 122 −43.555 15.861 12.003 0.50 55.52 C ATOM 1636 CD AGLU B 122 −42.272 16.342 11.353 0.50 61.34 C ATOM 1637 OE1 AGLU B 122 −41.715 17.363 11.819 0.50 43.76 O ATOM 1638 OE2 AGLU B 122 −41.822 15.699 10.379 0.50 49.62 O ATOM 1639 N BGLU B 122 −42.864 17.408 14.539 0.50 48.39 N ATOM 1640 CA BGLU B 122 −43.791 16.274 14.564 0.50 47.80 C ATOM 1641 C BGLU B 122 −43.703 15.544 15.913 0.50 49.98 C ATOM 1642 O BGLU B 122 −44.740 15.234 16.497 0.50 50.33 O ATOM 1643 CB BGLU B 122 −43.486 15.294 13.414 0.50 47.88 C ATOM 1644 CG BGLU B 122 −44.538 14.210 13.232 0.50 52.69 C ATOM 1645 CD BGLU B 122 −44.305 13.277 12.061 0.50 64.91 C ATOM 1646 OE1 BGLU B 122 −43.199 13.317 11.474 0.50 52.72 O ATOM 1647 OE2 BGLU B 122 −45.226 12.492 11.739 0.50 61.22 O ATOM 1648 N GLN B 123 −42.475 15.278 16.402 1.00 45.06 N ATOM 1649 CA GLN B 123 −42.270 14.580 17.678 1.00 44.94 C ATOM 1650 C GLN B 123 −42.759 15.406 18.858 1.00 50.36 C ATOM 1651 O GLN B 123 −43.385 14.847 19.762 1.00 49.32 O ATOM 1652 CB GLN B 123 −40.796 14.206 17.902 1.00 44.97 C ATOM 1653 CG GLN B 123 −40.619 13.279 19.109 1.00 44.15 C ATOM 1654 CD GLN B 123 −39.195 12.935 19.425 1.00 47.60 C ATOM 1655 OE1 GLN B 123 −38.258 13.662 19.082 1.00 40.87 O ATOM 1656 NE2 GLN B 123 −39.013 11.867 20.187 1.00 35.78 N ATOM 1657 N LEU B 124 −42.482 16.722 18.848 1.00 48.30 N ATOM 1658 CA LEU B 124 −42.917 17.624 19.909 1.00 50.36 C ATOM 1659 C LEU B 124 −44.442 17.657 20.030 1.00 57.38 C ATOM 1660 O LEU B 124 −44.938 17.783 21.146 1.00 58.17 O ATOM 1661 CB LEU B 124 −42.358 19.042 19.688 1.00 50.61 C ATOM 1662 CG LEU B 124 −40.841 19.179 19.840 1.00 54.63 C ATOM 1663 CD1 LEU B 124 −40.359 20.491 19.260 1.00 54.80 C ATOM 1664 CD2 LEU B 124 −40.419 19.070 21.288 1.00 57.16 C ATOM 1665 N LYS B 125 −45.189 17.457 18.916 1.00 54.79 N ATOM 1666 CA LYS B 125 −46.656 17.407 18.967 1.00 55.96 C ATOM 1667 C LYS B 125 −47.149 16.200 19.817 1.00 57.32 C ATOM 1668 O LYS B 125 −48.211 16.288 20.432 1.00 59.10 O ATOM 1669 CB LYS B 125 −47.261 17.374 17.549 1.00 59.50 C ATOM 1670 CG LYS B 125 −48.772 17.633 17.519 1.00 82.76 C ATOM 1671 CD LYS B 125 −49.194 18.695 16.493 1.00 95.44 C ATOM 1672 CE LYS B 125 −49.106 18.211 15.065 1.00 104.43 C ATOM 1673 NZ LYS B 125 −49.641 19.222 14.112 1.00 113.34 N ATOM 1674 N SER B 126 −46.357 15.112 19.890 1.00 50.44 N ATOM 1675 CA SER B 126 −46.663 13.934 20.718 1.00 49.24 C ATOM 1676 C SER B 126 −46.230 14.108 22.209 1.00 53.01 C ATOM 1677 O SER B 126 −46.458 13.198 23.015 1.00 51.52 O ATOM 1678 CB SER B 126 −46.032 12.678 20.119 1.00 49.01 C ATOM 1679 OG SER B 126 −44.662 12.539 20.458 1.00 53.59 O ATOM 1680 N GLY B 127 −45.596 15.254 22.560 1.00 51.44 N ATOM 1681 CA GLY B 127 −45.198 15.597 23.932 1.00 52.67 C ATOM 1682 C GLY B 127 −43.856 15.027 24.410 1.00 54.98 C ATOM 1683 O GLY B 127 −43.618 14.986 25.615 1.00 56.14 O ATOM 1684 N THR B 128 −42.966 14.666 23.479 1.00 49.82 N ATOM 1685 CA THR B 128 −41.624 14.131 23.754 1.00 48.90 C ATOM 1686 C THR B 128 −40.599 14.858 22.875 1.00 50.82 C ATOM 1687 O THR B 128 −40.967 15.427 21.846 1.00 48.54 O ATOM 1688 CB THR B 128 −41.577 12.640 23.439 1.00 55.72 C ATOM 1689 OG1 THR B 128 −42.047 12.433 22.100 1.00 52.09 O ATOM 1690 CG2 THR B 128 −42.389 11.825 24.407 1.00 55.78 C ATOM 1691 N ALA B 129 −39.318 14.815 23.264 1.00 47.60 N ATOM 1692 CA ALA B 129 −38.259 15.497 22.523 1.00 46.69 C ATOM 1693 C ALA B 129 −36.966 14.717 22.577 1.00 50.52 C ATOM 1694 O ALA B 129 −36.382 14.585 23.650 1.00 52.29 O ATOM 1695 CB ALA B 129 −38.043 16.889 23.103 1.00 48.80 C ATOM 1696 N SER B 130 −36.518 14.203 21.416 1.00 44.66 N ATOM 1697 CA SER B 130 −35.260 13.478 21.268 1.00 42.46 C ATOM 1698 C SER B 130 −34.256 14.415 20.612 1.00 45.91 C ATOM 1699 O SER B 130 −34.575 15.046 19.603 1.00 45.83 O ATOM 1700 CB SER B 130 −35.440 12.256 20.380 1.00 42.20 C ATOM 1701 OG SER B 130 −36.417 11.377 20.904 1.00 42.28 O ATOM 1702 N VAL B 131 −33.057 14.520 21.184 1.00 41.62 N ATOM 1703 CA VAL B 131 −31.980 15.360 20.659 1.00 39.96 C ATOM 1704 C VAL B 131 −30.892 14.375 20.300 1.00 40.80 C ATOM 1705 O VAL B 131 −30.533 13.549 21.143 1.00 39.10 O ATOM 1706 CB VAL B 131 −31.480 16.397 21.698 1.00 45.31 C ATOM 1707 CG1 VAL B 131 −30.610 17.444 21.022 1.00 45.09 C ATOM 1708 CG2 VAL B 131 −32.645 17.070 22.422 1.00 45.92 C ATOM 1709 N VAL B 132 −30.429 14.392 19.040 1.00 38.45 N ATOM 1710 CA VAL B 132 −29.446 13.429 18.543 1.00 37.72 C ATOM 1711 C VAL B 132 −28.118 14.116 18.237 1.00 43.59 C ATOM 1712 O VAL B 132 −28.091 15.139 17.553 1.00 40.69 O ATOM 1713 CB VAL B 132 −29.994 12.652 17.308 1.00 39.35 C ATOM 1714 CG1 VAL B 132 −28.916 11.770 16.662 1.00 37.62 C ATOM 1715 CG2 VAL B 132 −31.214 11.812 17.691 1.00 38.39 C ATOM 1716 N CYS B 133 −27.022 13.516 18.726 1.00 43.57 N ATOM 1717 CA CYS B 133 −25.663 13.933 18.441 1.00 45.44 C ATOM 1718 C CYS B 133 −25.055 12.811 17.612 1.00 45.60 C ATOM 1719 O CYS B 133 −25.119 11.649 18.026 1.00 43.13 O ATOM 1720 CB CYS B 133 −24.872 14.159 19.721 1.00 49.38 C ATOM 1721 SG CYS B 133 −23.190 14.764 19.439 1.00 56.38 S ATOM 1722 N LEU B 134 −24.495 13.142 16.438 1.00 41.51 N ATOM 1723 CA LEU B 134 −23.869 12.158 15.544 1.00 39.97 C ATOM 1724 C LEU B 134 −22.366 12.407 15.505 1.00 44.72 C ATOM 1725 O LEU B 134 −21.950 13.543 15.305 1.00 44.75 O ATOM 1726 CB LEU B 134 −24.470 12.265 14.124 1.00 38.04 C ATOM 1727 CG LEU B 134 −23.730 11.552 12.969 1.00 40.51 C ATOM 1728 CD1 LEU B 134 −23.798 10.045 13.114 1.00 38.88 C ATOM 1729 CD2 LEU B 134 −24.311 11.972 11.627 1.00 40.63 C ATOM 1730 N LEU B 135 −21.565 11.345 15.704 1.00 41.00 N ATOM 1731 CA LEU B 135 −20.098 11.363 15.605 1.00 41.97 C ATOM 1732 C LEU B 135 −19.822 10.484 14.404 1.00 44.74 C ATOM 1733 O LEU B 135 −20.012 9.274 14.496 1.00 43.15 O ATOM 1734 CB LEU B 135 −19.441 10.746 16.840 1.00 42.81 C ATOM 1735 CG LEU B 135 −19.313 11.619 18.065 1.00 47.57 C ATOM 1736 CD1 LEU B 135 −20.682 11.976 18.646 1.00 45.53 C ATOM 1737 CD2 LEU B 135 −18.423 10.926 19.092 1.00 52.20 C ATOM 1738 N ASN B 136 −19.447 11.078 13.266 1.00 42.96 N ATOM 1739 CA ASN B 136 −19.294 10.330 12.020 1.00 42.15 C ATOM 1740 C ASN B 136 −17.857 10.054 11.580 1.00 47.55 C ATOM 1741 O ASN B 136 −17.021 10.958 11.585 1.00 49.26 O ATOM 1742 CB ASN B 136 −20.029 11.085 10.904 1.00 41.37 C ATOM 1743 CG ASN B 136 −20.616 10.190 9.844 1.00 51.90 C ATOM 1744 OD1 ASN B 136 −21.276 9.205 10.146 1.00 44.17 O ATOM 1745 ND2 ASN B 136 −20.409 10.507 8.581 1.00 47.56 N ATOM 1746 N ASN B 137 −17.602 8.804 11.148 1.00 43.79 N ATOM 1747 CA ASN B 137 −16.358 8.338 10.532 1.00 45.11 C ATOM 1748 C ASN B 137 −15.088 8.625 11.325 1.00 50.60 C ATOM 1749 O ASN B 137 −14.222 9.361 10.858 1.00 52.71 O ATOM 1750 CB ASN B 137 −16.248 8.922 9.112 1.00 47.23 C ATOM 1751 CG ASN B 137 −17.433 8.599 8.231 1.00 61.25 C ATOM 1752 OD1 ASN B 137 −18.151 7.618 8.456 1.00 50.10 O ATOM 1753 ND2 ASN B 137 −17.680 9.421 7.214 1.00 51.12 N ATOM 1754 N PHE B 138 −14.945 7.980 12.488 1.00 46.69 N ATOM 1755 CA PHE B 138 −13.786 8.163 13.358 1.00 48.08 C ATOM 1756 C PHE B 138 −13.085 6.847 13.723 1.00 51.74 C ATOM 1757 O PHE B 138 −13.678 5.771 13.650 1.00 49.10 O ATOM 1758 CB PHE B 138 −14.216 8.908 14.630 1.00 49.40 C ATOM 1759 CG PHE B 138 −15.186 8.155 15.508 1.00 48.95 C ATOM 1760 CD1 PHE B 138 −14.729 7.316 16.511 1.00 52.66 C ATOM 1761 CD2 PHE B 138 −16.557 8.273 15.321 1.00 48.13 C ATOM 1762 CE1 PHE B 138 −15.619 6.613 17.321 1.00 51.64 C ATOM 1763 CE2 PHE B 138 −17.449 7.570 16.135 1.00 49.29 C ATOM 1764 CZ PHE B 138 −16.974 6.755 17.136 1.00 48.25 C ATOM 1765 N TYR B 139 −11.817 6.959 14.130 1.00 51.54 N ATOM 1766 CA TYR B 139 −10.994 5.830 14.574 1.00 52.33 C ATOM 1767 C TYR B 139 −9.969 6.356 15.622 1.00 57.69 C ATOM 1768 O TYR B 139 −9.383 7.406 15.374 1.00 57.85 O ATOM 1769 CB TYR B 139 −10.264 5.159 13.385 1.00 54.15 C ATOM 1770 CG TYR B 139 −9.572 3.896 13.834 1.00 56.42 C ATOM 1771 CD1 TYR B 139 −10.269 2.697 13.924 1.00 55.93 C ATOM 1772 CD2 TYR B 139 −8.299 3.945 14.397 1.00 59.79 C ATOM 1773 CE1 TYR B 139 −9.700 1.567 14.503 1.00 56.81 C ATOM 1774 CE2 TYR B 139 −7.735 2.831 15.012 1.00 61.40 C ATOM 1775 CZ TYR B 139 −8.426 1.633 15.036 1.00 66.36 C ATOM 1776 OH TYR B 139 −7.844 0.526 15.609 1.00 67.92 O ATOM 1777 N PRO B 140 −9.736 5.656 16.768 1.00 54.87 N ATOM 1778 CA PRO B 140 −10.313 4.383 17.238 1.00 52.80 C ATOM 1779 C PRO B 140 −11.744 4.520 17.769 1.00 56.42 C ATOM 1780 O PRO B 140 −12.290 5.621 17.803 1.00 55.80 O ATOM 1781 CB PRO B 140 −9.315 3.940 18.314 1.00 56.32 C ATOM 1782 CG PRO B 140 −8.826 5.225 18.901 1.00 62.48 C ATOM 1783 CD PRO B 140 −8.750 6.185 17.735 1.00 58.28 C ATOM 1784 N ARG B 141 −12.342 3.389 18.183 1.00 52.96 N ATOM 1785 CA ARG B 141 −13.720 3.330 18.693 1.00 51.71 C ATOM 1786 C ARG B 141 −13.925 4.112 19.999 1.00 57.68 C ATOM 1787 O ARG B 141 −15.039 4.571 20.265 1.00 55.60 O ATOM 1788 CB ARG B 141 −14.138 1.862 18.916 1.00 50.28 C ATOM 1789 CG ARG B 141 −15.639 1.653 19.145 1.00 57.39 C ATOM 1790 CD ARG B 141 −15.895 1.022 20.500 1.00 77.69 C ATOM 1791 NE ARG B 141 −17.298 0.667 20.723 1.00 85.89 N ATOM 1792 CZ ARG B 141 −17.925 −0.385 20.195 1.00 91.53 C ATOM 1793 NH1 ARG B 141 −17.288 −1.202 19.358 1.00 73.57 N ATOM 1794 NH2 ARG B 141 −19.200 −0.618 20.484 1.00 73.45 N ATOM 1795 N GLU B 142 −12.866 4.260 20.810 1.00 57.75 N ATOM 1796 CA GLU B 142 −12.949 4.945 22.103 1.00 59.18 C ATOM 1797 C GLU B 142 −13.297 6.418 21.896 1.00 64.31 C ATOM 1798 O GLU B 142 −12.537 7.142 21.260 1.00 65.59 O ATOM 1799 CB GLU B 142 −11.636 4.801 22.903 1.00 63.33 C ATOM 1800 CG GLU B 142 −11.400 3.400 23.459 1.00 70.25 C ATOM 1801 CD GLU B 142 −11.058 2.319 22.450 1.00 79.12 C ATOM 1802 OE1 GLU B 142 −10.233 2.590 21.547 1.00 62.08 O ATOM 1803 OE2 GLU B 142 −11.634 1.210 22.544 1.00 65.27 O ATOM 1804 N ALA B 143 −14.482 6.830 22.375 1.00 60.05 N ATOM 1805 CA ALA B 143 −14.992 8.195 22.251 1.00 60.25 C ATOM 1806 C ALA B 143 −15.937 8.492 23.413 1.00 64.55 C ATOM 1807 O ALA B 143 −16.697 7.613 23.808 1.00 63.46 O ATOM 1808 CB ALA B 143 −15.748 8.343 20.935 1.00 58.79 C ATOM 1809 N LYS B 144 −15.902 9.717 23.954 1.00 63.21 N ATOM 1810 CA LYS B 144 −16.783 10.102 25.057 1.00 63.15 C ATOM 1811 C LYS B 144 −17.698 11.234 24.606 1.00 64.62 C ATOM 1812 O LYS B 144 −17.220 12.234 24.074 1.00 64.21 O ATOM 1813 CB LYS B 144 −15.965 10.524 26.288 1.00 68.96 C ATOM 1814 CG LYS B 144 −16.710 10.327 27.611 1.00 89.48 C ATOM 1815 CD LYS B 144 −16.363 9.010 28.347 1.00 101.00 C ATOM 1816 CE LYS B 144 −17.045 7.757 27.817 1.00 107.47 C ATOM 1817 NZ LYS B 144 −16.234 6.991 26.826 1.00 113.21 N ATOM 1818 N VAL B 145 −19.015 11.060 24.803 1.00 58.70 N ATOM 1819 CA VAL B 145 −20.027 12.058 24.465 1.00 56.79 C ATOM 1820 C VAL B 145 −20.631 12.553 25.776 1.00 61.26 C ATOM 1821 O VAL B 145 −20.996 11.734 26.627 1.00 61.28 O ATOM 1822 CB VAL B 145 −21.111 11.474 23.537 1.00 57.49 C ATOM 1823 CG1 VAL B 145 −22.171 12.525 23.217 1.00 56.36 C ATOM 1824 CG2 VAL B 145 −20.485 10.928 22.259 1.00 56.26 C ATOM 1825 N GLN B 146 −20.742 13.884 25.934 1.00 57.91 N ATOM 1826 CA GLN B 146 −21.286 14.498 27.141 1.00 58.76 C ATOM 1827 C GLN B 146 −22.366 15.513 26.749 1.00 60.79 C ATOM 1828 O GLN B 146 −22.095 16.424 25.970 1.00 60.97 O ATOM 1829 CB GLN B 146 −20.138 15.151 27.937 1.00 63.02 C ATOM 1830 CG GLN B 146 −20.525 15.781 29.273 1.00 81.67 C ATOM 1831 CD GLN B 146 −21.055 14.769 30.256 1.00 101.31 C ATOM 1832 OE1 GLN B 146 −20.292 14.067 30.923 1.00 99.91 O ATOM 1833 NE2 GLN B 146 −22.371 14.695 30.410 1.00 90.85 N ATOM 1834 N TRP B 147 −23.594 15.322 27.255 1.00 55.77 N ATOM 1835 CA TRP B 147 −24.729 16.209 26.985 1.00 54.82 C ATOM 1836 C TRP B 147 −24.811 17.330 28.040 1.00 61.88 C ATOM 1837 O TRP B 147 −24.635 17.056 29.229 1.00 63.01 O ATOM 1838 CB TRP B 147 −26.055 15.413 26.994 1.00 51.74 C ATOM 1839 CG TRP B 147 −26.329 14.639 25.743 1.00 50.01 C ATOM 1840 CD1 TRP B 147 −26.211 13.293 25.565 1.00 51.63 C ATOM 1841 CD2 TRP B 147 −26.789 15.172 24.497 1.00 48.35 C ATOM 1842 NE1 TRP B 147 −26.571 12.953 24.281 1.00 48.77 N ATOM 1843 CE2 TRP B 147 −26.909 14.092 23.597 1.00 50.07 C ATOM 1844 CE3 TRP B 147 −27.063 16.468 24.037 1.00 49.60 C ATOM 1845 CZ2 TRP B 147 −27.342 14.265 22.280 1.00 47.70 C ATOM 1846 CZ3 TRP B 147 −27.501 16.634 22.736 1.00 49.20 C ATOM 1847 CH2 TRP B 147 −27.636 15.541 21.872 1.00 48.13 C ATOM 1848 N LYS B 148 −25.130 18.566 27.610 1.00 58.30 N ATOM 1849 CA LYS B 148 −25.332 19.712 28.510 1.00 60.71 C ATOM 1850 C LYS B 148 −26.619 20.443 28.111 1.00 65.11 C ATOM 1851 O LYS B 148 −26.851 20.668 26.921 1.00 63.98 O ATOM 1852 CB LYS B 148 −24.155 20.703 28.479 1.00 63.88 C ATOM 1853 CG LYS B 148 −22.779 20.072 28.617 1.00 77.60 C ATOM 1854 CD LYS B 148 −21.693 21.133 28.765 1.00 88.29 C ATOM 1855 CE LYS B 148 −20.333 20.627 28.351 1.00 99.31 C ATOM 1856 NZ LYS B 148 −19.256 21.605 28.660 1.00 112.28 N ATOM 1857 N VAL B 149 −27.450 20.799 29.105 1.00 62.45 N ATOM 1858 CA VAL B 149 −28.714 21.515 28.916 1.00 61.78 C ATOM 1859 C VAL B 149 −28.569 22.808 29.737 1.00 66.99 C ATOM 1860 O VAL B 149 −28.625 22.746 30.965 1.00 67.90 O ATOM 1861 CB VAL B 149 −29.905 20.624 29.360 1.00 65.16 C ATOM 1862 CG1 VAL B 149 −31.217 21.396 29.334 1.00 65.00 C ATOM 1863 CG2 VAL B 149 −30.003 19.375 28.480 1.00 62.45 C ATOM 1864 N ASP B 150 −28.293 23.951 29.059 1.00 63.79 N ATOM 1865 CA ASP B 150 −27.964 25.252 29.682 1.00 66.94 C ATOM 1866 C ASP B 150 −26.665 25.100 30.502 1.00 76.26 C ATOM 1867 O ASP B 150 −26.588 25.507 31.664 1.00 78.68 O ATOM 1868 CB ASP B 150 −29.124 25.835 30.533 1.00 69.01 C ATOM 1869 CG ASP B 150 −30.327 26.308 29.741 1.00 71.79 C ATOM 1870 OD1 ASP B 150 −30.173 26.595 28.530 1.00 70.87 O ATOM 1871 OD2 ASP B 150 −31.419 26.423 30.336 1.00 74.21 O ATOM 1872 N ASN B 151 −25.658 24.442 29.883 1.00 74.05 N ATOM 1873 CA ASN B 151 −24.336 24.155 30.457 1.00 76.87 C ATOM 1874 C ASN B 151 −24.361 23.221 31.701 1.00 83.21 C ATOM 1875 O ASN B 151 −23.322 23.066 32.348 1.00 85.06 O ATOM 1876 CB ASN B 151 −23.579 25.462 30.764 1.00 82.11 C ATOM 1877 CG ASN B 151 −22.138 25.425 30.321 1.00 113.45 C ATOM 1878 OD1 ASN B 151 −21.777 25.941 29.256 1.00 105.66 O ATOM 1879 ND2 ASN B 151 −21.294 24.762 31.101 1.00 110.93 N ATOM 1880 N ALA B 152 −25.511 22.568 32.002 1.00 78.34 N ATOM 1881 CA ALA B 152 −25.645 21.670 33.147 1.00 78.90 C ATOM 1882 C ALA B 152 −25.420 20.232 32.672 1.00 81.83 C ATOM 1883 O ALA B 152 −26.232 19.696 31.913 1.00 78.13 O ATOM 1884 CB ALA B 152 −27.028 21.809 33.771 1.00 79.41 C ATOM 1885 N LEU B 153 −24.301 19.625 33.113 1.00 80.85 N ATOM 1886 CA LEU B 153 −23.903 18.248 32.772 1.00 79.40 C ATOM 1887 C LEU B 153 −25.063 17.260 32.979 1.00 80.64 C ATOM 1888 O LEU B 153 −25.703 17.290 34.031 1.00 81.20 O ATOM 1889 CB LEU B 153 −22.686 17.812 33.622 1.00 82.41 C ATOM 1890 CG LEU B 153 −21.589 17.104 32.849 1.00 87.34 C ATOM 1891 CD1 LEU B 153 −20.707 18.102 32.106 1.00 88.51 C ATOM 1892 CD2 LEU B 153 −20.736 16.229 33.765 1.00 92.60 C ATOM 1893 N GLN B 154 −25.344 16.410 31.973 1.00 74.56 N ATOM 1894 CA GLN B 154 −26.447 15.443 32.030 1.00 72.41 C ATOM 1895 C GLN B 154 −25.960 14.033 32.365 1.00 76.58 C ATOM 1896 O GLN B 154 −24.771 13.734 32.220 1.00 76.89 O ATOM 1897 CB GLN B 154 −27.204 15.429 30.692 1.00 70.53 C ATOM 1898 CG GLN B 154 −27.877 16.755 30.338 1.00 75.29 C ATOM 1899 CD GLN B 154 −29.096 17.045 31.190 1.00 85.67 C ATOM 1900 OE1 GLN B 154 −30.108 16.341 31.118 1.00 78.23 O ATOM 1901 NE2 GLN B 154 −29.056 18.105 31.993 1.00 79.95 N ATOM 1902 N SER B 155 −26.891 13.167 32.816 1.00 72.43 N ATOM 1903 CA SER B 155 −26.583 11.771 33.149 1.00 71.75 C ATOM 1904 C SER B 155 −27.849 10.909 33.296 1.00 73.00 C ATOM 1905 O SER B 155 −28.813 11.331 33.942 1.00 72.41 O ATOM 1906 CB SER B 155 −25.758 11.695 34.435 1.00 79.51 C ATOM 1907 OG SER B 155 −24.402 11.392 34.151 1.00 91.92 O ATOM 1908 N GLY B 156 −27.826 9.692 32.704 1.00 67.51 N ATOM 1909 CA GLY B 156 −28.912 8.717 32.819 1.00 65.86 C ATOM 1910 C GLY B 156 −30.145 8.940 31.943 1.00 67.02 C ATOM 1911 O GLY B 156 −31.125 8.218 32.123 1.00 66.32 O ATOM 1912 N ASN B 157 −30.118 9.907 31.008 1.00 62.63 N ATOM 1913 CA ASN B 157 −31.252 10.154 30.101 1.00 60.49 C ATOM 1914 C ASN B 157 −30.831 10.095 28.615 1.00 61.02 C ATOM 1915 O ASN B 157 −31.612 10.498 27.748 1.00 59.01 O ATOM 1916 CB ASN B 157 −31.960 11.480 30.454 1.00 60.32 C ATOM 1917 CG ASN B 157 −31.079 12.704 30.469 1.00 74.28 C ATOM 1918 OD1 ASN B 157 −29.851 12.635 30.290 1.00 59.89 O ATOM 1919 ND2 ASN B 157 −31.692 13.857 30.718 1.00 67.62 N ATOM 1920 N SER B 158 −29.620 9.551 28.325 1.00 55.90 N ATOM 1921 CA SER B 158 −29.101 9.406 26.967 1.00 53.60 C ATOM 1922 C SER B 158 −28.739 7.946 26.689 1.00 56.48 C ATOM 1923 O SER B 158 −28.461 7.193 27.621 1.00 57.31 O ATOM 1924 CB SER B 158 −27.880 10.295 26.752 1.00 56.49 C ATOM 1925 OG SER B 158 −26.738 9.800 27.431 1.00 63.69 O ATOM 1926 N GLN B 159 −28.744 7.555 25.409 1.00 49.85 N ATOM 1927 CA GLN B 159 −28.380 6.204 24.990 1.00 48.14 C ATOM 1928 C GLN B 159 −27.501 6.279 23.760 1.00 48.42 C ATOM 1929 O GLN B 159 −27.720 7.117 22.882 1.00 46.22 O ATOM 1930 CB GLN B 159 −29.618 5.333 24.746 1.00 48.47 C ATOM 1931 CG GLN B 159 −30.077 4.623 26.008 1.00 60.61 C ATOM 1932 CD GLN B 159 −31.061 3.531 25.698 1.00 74.74 C ATOM 1933 OE1 GLN B 159 −31.915 3.676 24.823 1.00 65.28 O ATOM 1934 NE2 GLN B 159 −30.957 2.403 26.391 1.00 69.75 N ATOM 1935 N GLU B 160 −26.495 5.413 23.723 1.00 43.16 N ATOM 1936 CA GLU B 160 −25.484 5.376 22.688 1.00 42.59 C ATOM 1937 C GLU B 160 −25.624 4.142 21.805 1.00 44.01 C ATOM 1938 O GLU B 160 −26.049 3.083 22.277 1.00 40.63 O ATOM 1939 CB GLU B 160 −24.101 5.372 23.360 1.00 46.21 C ATOM 1940 CG GLU B 160 −23.067 6.266 22.717 1.00 59.67 C ATOM 1941 CD GLU B 160 −22.110 6.871 23.721 1.00 82.32 C ATOM 1942 OE1 GLU B 160 −22.489 7.872 24.371 1.00 76.02 O ATOM 1943 OE2 GLU B 160 −20.996 6.323 23.882 1.00 81.97 O ATOM 1944 N SER B 161 −25.250 4.289 20.522 1.00 40.24 N ATOM 1945 CA SER B 161 −25.218 3.192 19.555 1.00 39.43 C ATOM 1946 C SER B 161 −24.028 3.427 18.640 1.00 43.55 C ATOM 1947 O SER B 161 −23.806 4.559 18.232 1.00 42.87 O ATOM 1948 CB SER B 161 −26.502 3.125 18.740 1.00 41.55 C ATOM 1949 OG SER B 161 −26.503 1.948 17.952 1.00 46.47 O ATOM 1950 N VAL B 162 −23.255 2.375 18.342 1.00 40.28 N ATOM 1951 CA VAL B 162 −22.059 2.460 17.501 1.00 40.87 C ATOM 1952 C VAL B 162 −22.214 1.456 16.365 1.00 45.52 C ATOM 1953 O VAL B 162 −22.662 0.336 16.602 1.00 44.96 O ATOM 1954 CB VAL B 162 −20.769 2.153 18.308 1.00 45.94 C ATOM 1955 CG1 VAL B 162 −19.534 2.668 17.582 1.00 46.43 C ATOM 1956 CG2 VAL B 162 −20.845 2.737 19.715 1.00 47.46 C ATOM 1957 N THR B 163 −21.814 1.834 15.146 1.00 42.14 N ATOM 1958 CA THR B 163 −21.870 0.920 14.011 1.00 40.76 C ATOM 1959 C THR B 163 −20.678 −0.015 14.098 1.00 46.05 C ATOM 1960 O THR B 163 −19.743 0.236 14.866 1.00 46.24 O ATOM 1961 CB THR B 163 −21.794 1.696 12.670 1.00 44.21 C ATOM 1962 OG1 THR B 163 −20.598 2.475 12.635 1.00 42.58 O ATOM 1963 CG2 THR B 163 −22.983 2.594 12.448 1.00 39.52 C ATOM 1964 N GLU B 164 −20.681 −1.064 13.258 1.00 42.71 N ATOM 1965 CA GLU B 164 −19.534 −1.959 13.127 1.00 43.35 C ATOM 1966 C GLU B 164 −18.502 −1.185 12.300 1.00 48.75 C ATOM 1967 O GLU B 164 −18.853 −0.214 11.630 1.00 47.54 O ATOM 1968 CB GLU B 164 −19.905 −3.258 12.382 1.00 43.58 C ATOM 1969 CG GLU B 164 −20.941 −4.126 13.070 1.00 47.89 C ATOM 1970 CD GLU B 164 −20.526 −4.753 14.386 1.00 69.07 C ATOM 1971 OE1 GLU B 164 −19.318 −4.727 14.717 1.00 62.05 O ATOM 1972 OE2 GLU B 164 −21.417 −5.296 15.078 1.00 66.26 O ATOM 1973 N GLN B 165 −17.250 −1.629 12.315 1.00 48.38 N ATOM 1974 CA GLN B 165 −16.171 −0.980 11.560 1.00 49.62 C ATOM 1975 C GLN B 165 −16.509 −1.009 10.055 1.00 55.31 C ATOM 1976 O GLN B 165 −16.901 −2.058 9.546 1.00 55.76 O ATOM 1977 CB GLN B 165 −14.849 −1.691 11.865 1.00 52.40 C ATOM 1978 CG GLN B 165 −13.627 −0.826 11.689 1.00 60.35 C ATOM 1979 CD GLN B 165 −12.393 −1.435 12.293 1.00 67.84 C ATOM 1980 OE1 GLN B 165 −12.333 −2.632 12.593 1.00 56.87 O ATOM 1981 NE2 GLN B 165 −11.353 −0.635 12.425 1.00 63.60 N ATOM 1982 N ASP B 166 −16.427 0.145 9.366 1.00 53.07 N ATOM 1983 CA ASP B 166 −16.826 0.258 7.955 1.00 53.01 C ATOM 1984 C ASP B 166 −16.078 −0.676 6.977 1.00 59.36 C ATOM 1985 O ASP B 166 −14.854 −0.792 7.029 1.00 60.34 O ATOM 1986 CB ASP B 166 −16.709 1.704 7.468 1.00 55.20 C ATOM 1987 CG ASP B 166 −17.418 1.949 6.151 1.00 59.71 C ATOM 1988 OD1 ASP B 166 −18.669 1.950 6.142 1.00 59.68 O ATOM 1989 OD2 ASP B 166 −16.722 2.130 5.129 1.00 59.98 O ATOM 1990 N SER B 167 −16.835 −1.292 6.050 1.00 56.31 N ATOM 1991 CA SER B 167 −16.315 −2.227 5.049 1.00 57.19 C ATOM 1992 C SER B 167 −15.273 −1.608 4.105 1.00 61.95 C ATOM 1993 O SER B 167 −14.359 −2.317 3.690 1.00 62.89 O ATOM 1994 CB SER B 167 −17.466 −2.827 4.244 1.00 60.76 C ATOM 1995 OG SER B 167 −18.241 −1.811 3.629 1.00 73.16 O ATOM 1996 N LYS B 168 −15.388 −0.293 3.791 1.00 58.37 N ATOM 1997 CA LYS B 168 −14.447 0.403 2.905 1.00 59.84 C ATOM 1998 C LYS B 168 −13.346 1.174 3.653 1.00 63.38 C ATOM 1999 O LYS B 168 −12.168 0.911 3.422 1.00 64.86 O ATOM 2000 CB LYS B 168 −15.198 1.351 1.946 1.00 62.47 C ATOM 2001 CG LYS B 168 −14.269 2.264 1.131 1.00 83.02 C ATOM 2002 CD LYS B 168 −14.900 2.799 −0.158 1.00 94.89 C ATOM 2003 CE LYS B 168 −14.596 4.264 −0.391 1.00 110.05 C ATOM 2004 NZ LYS B 168 −14.582 4.617 −1.832 1.00 121.69 N ATOM 2005 N ASP B 169 −13.724 2.163 4.483 1.00 58.13 N ATOM 2006 CA ASP B 169 −12.754 3.035 5.171 1.00 58.45 C ATOM 2007 C ASP B 169 −12.375 2.593 6.608 1.00 58.86 C ATOM 2008 O ASP B 169 −11.598 3.297 7.226 1.00 59.24 O ATOM 2009 CB ASP B 169 −13.230 4.514 5.153 1.00 59.93 C ATOM 2010 CG ASP B 169 −14.593 4.792 5.768 1.00 66.31 C ATOM 2011 OD1 ASP B 169 −14.877 4.252 6.856 1.00 65.52 O ATOM 2012 OD2 ASP B 169 −15.355 5.594 5.187 1.00 67.95 O ATOM 2013 N SER B 170 −12.892 1.457 7.130 1.00 52.83 N ATOM 2014 CA SER B 170 −12.551 0.920 8.468 1.00 51.74 C ATOM 2015 C SER B 170 −12.756 1.893 9.683 1.00 54.52 C ATOM 2016 O SER B 170 −12.112 1.713 10.719 1.00 54.26 O ATOM 2017 CB SER B 170 −11.121 0.376 8.475 1.00 56.38 C ATOM 2018 OG SER B 170 −10.912 −0.598 7.465 1.00 61.79 O ATOM 2019 N THR B 171 −13.678 2.870 9.581 1.00 50.06 N ATOM 2020 CA THR B 171 −13.979 3.788 10.688 1.00 49.35 C ATOM 2021 C THR B 171 −15.274 3.377 11.383 1.00 51.64 C ATOM 2022 O THR B 171 −16.048 2.563 10.867 1.00 49.92 O ATOM 2023 CB THR B 171 −14.165 5.245 10.192 1.00 53.05 C ATOM 2024 OG1 THR B 171 −15.281 5.301 9.296 1.00 45.64 O ATOM 2025 CG2 THR B 171 −12.905 5.831 9.567 1.00 51.38 C ATOM 2026 N TYR B 172 −15.518 4.001 12.534 1.00 48.20 N ATOM 2027 CA TYR B 172 −16.731 3.847 13.322 1.00 46.06 C ATOM 2028 C TYR B 172 −17.550 5.127 13.218 1.00 48.04 C ATOM 2029 O TYR B 172 −17.003 6.203 12.962 1.00 47.47 O ATOM 2030 CB TYR B 172 −16.375 3.648 14.800 1.00 47.86 C ATOM 2031 CG TYR B 172 −15.664 2.347 15.070 1.00 50.29 C ATOM 2032 CD1 TYR B 172 −14.281 2.249 14.961 1.00 54.19 C ATOM 2033 CD2 TYR B 172 −16.373 1.207 15.436 1.00 49.40 C ATOM 2034 CE1 TYR B 172 −13.622 1.046 15.205 1.00 56.13 C ATOM 2035 CE2 TYR B 172 −15.726 0.002 15.687 1.00 50.21 C ATOM 2036 CZ TYR B 172 −14.353 −0.080 15.553 1.00 58.65 C ATOM 2037 OH TYR B 172 −13.732 −1.266 15.836 1.00 57.48 O ATOM 2038 N SER B 173 −18.853 5.005 13.449 1.00 42.52 N ATOM 2039 CA SER B 173 −19.781 6.128 13.554 1.00 41.39 C ATOM 2040 C SER B 173 −20.567 5.872 14.829 1.00 44.26 C ATOM 2041 O SER B 173 −20.687 4.717 15.243 1.00 42.74 O ATOM 2042 CB SER B 173 −20.688 6.231 12.336 1.00 42.46 C ATOM 2043 OG SER B 173 −19.968 6.785 11.247 1.00 48.33 O ATOM 2044 N LEU B 174 −21.067 6.926 15.473 1.00 41.73 N ATOM 2045 CA LEU B 174 −21.758 6.794 16.750 1.00 41.45 C ATOM 2046 C LEU B 174 −22.885 7.807 16.895 1.00 45.24 C ATOM 2047 O LEU B 174 −22.722 8.965 16.500 1.00 44.85 O ATOM 2048 CB LEU B 174 −20.711 6.948 17.870 1.00 43.33 C ATOM 2049 CG LEU B 174 −21.169 6.802 19.328 1.00 48.59 C ATOM 2050 CD1 LEU B 174 −20.045 6.224 20.192 1.00 50.45 C ATOM 2051 CD2 LEU B 174 −21.589 8.146 19.925 1.00 52.10 C ATOM 2052 N SER B 175 −24.022 7.377 17.494 1.00 40.43 N ATOM 2053 CA SER B 175 −25.159 8.257 17.785 1.00 39.27 C ATOM 2054 C SER B 175 −25.413 8.287 19.306 1.00 43.68 C ATOM 2055 O SER B 175 −25.371 7.237 19.955 1.00 44.00 O ATOM 2056 CB SER B 175 −26.419 7.749 17.098 1.00 39.91 C ATOM 2057 OG SER B 175 −26.842 6.540 17.710 1.00 46.16 O ATOM 2058 N SER B 176 −25.725 9.460 19.850 1.00 39.04 N ATOM 2059 CA SER B 176 −26.103 9.626 21.255 1.00 39.59 C ATOM 2060 C SER B 176 −27.460 10.313 21.229 1.00 43.39 C ATOM 2061 O SER B 176 −27.584 11.352 20.588 1.00 42.69 O ATOM 2062 CB SER B 176 −25.083 10.467 22.009 1.00 44.01 C ATOM 2063 OG SER B 176 −25.406 10.533 23.388 1.00 53.29 O ATOM 2064 N THR B 177 −28.494 9.694 21.813 1.00 39.57 N ATOM 2065 CA THR B 177 −29.853 10.234 21.800 1.00 38.89 C ATOM 2066 C THR B 177 −30.309 10.614 23.207 1.00 45.78 C ATOM 2067 O THR B 177 −30.455 9.739 24.061 1.00 46.82 O ATOM 2068 CB THR B 177 −30.820 9.242 21.152 1.00 42.72 C ATOM 2069 OG1 THR B 177 −30.292 8.833 19.885 1.00 41.05 O ATOM 2070 CG2 THR B 177 −32.216 9.833 20.964 1.00 40.93 C ATOM 2071 N LEU B 178 −30.578 11.912 23.423 1.00 44.32 N ATOM 2072 CA LEU B 178 −31.063 12.457 24.691 1.00 45.67 C ATOM 2073 C LEU B 178 −32.572 12.606 24.560 1.00 51.59 C ATOM 2074 O LEU B 178 −33.017 13.348 23.691 1.00 50.54 O ATOM 2075 CB LEU B 178 −30.421 13.829 24.939 1.00 46.50 C ATOM 2076 CG LEU B 178 −30.877 14.609 26.178 1.00 51.65 C ATOM 2077 CD1 LEU B 178 −30.287 14.020 27.432 1.00 52.40 C ATOM 2078 CD2 LEU B 178 −30.473 16.062 26.066 1.00 55.19 C ATOM 2079 N THR B 179 −33.359 11.906 25.400 1.00 50.94 N ATOM 2080 CA THR B 179 −34.823 11.978 25.341 1.00 51.02 C ATOM 2081 C THR B 179 −35.379 12.693 26.588 1.00 54.86 C ATOM 2082 O THR B 179 −35.180 12.233 27.712 1.00 54.95 O ATOM 2083 CB THR B 179 −35.427 10.576 25.091 1.00 62.65 C ATOM 2084 OG1 THR B 179 −34.903 10.049 23.857 1.00 67.28 O ATOM 2085 CG2 THR B 179 −36.950 10.604 24.996 1.00 60.77 C ATOM 2086 N LEU B 180 −36.077 13.813 26.374 1.00 50.66 N ATOM 2087 CA LEU B 180 −36.713 14.588 27.429 1.00 52.20 C ATOM 2088 C LEU B 180 −38.189 14.686 27.132 1.00 52.81 C ATOM 2089 O LEU B 180 −38.622 14.406 26.020 1.00 49.03 O ATOM 2090 CB LEU B 180 −36.147 16.020 27.458 1.00 53.77 C ATOM 2091 CG LEU B 180 −34.650 16.163 27.669 1.00 59.65 C ATOM 2092 CD1 LEU B 180 −34.262 17.619 27.663 1.00 61.07 C ATOM 2093 CD2 LEU B 180 −34.213 15.535 28.986 1.00 63.66 C ATOM 2094 N SER B 181 −38.962 15.152 28.107 1.00 52.27 N ATOM 2095 CA SER B 181 −40.374 15.437 27.886 1.00 52.01 C ATOM 2096 C SER B 181 −40.429 16.787 27.163 1.00 56.45 C ATOM 2097 O SER B 181 −39.449 17.538 27.208 1.00 55.99 O ATOM 2098 CB SER B 181 −41.117 15.518 29.217 1.00 55.17 C ATOM 2099 OG SER B 181 −40.678 16.608 30.012 1.00 55.33 O ATOM 2100 N LYS B 182 −41.550 17.091 26.483 1.00 53.85 N ATOM 2101 CA LYS B 182 −41.731 18.386 25.811 1.00 53.81 C ATOM 2102 C LYS B 182 −41.730 19.486 26.880 1.00 59.10 C ATOM 2103 O LYS B 182 −41.168 20.556 26.641 1.00 58.25 O ATOM 2104 CB LYS B 182 −43.045 18.404 25.009 1.00 55.66 C ATOM 2105 CG LYS B 182 −43.313 19.643 24.163 1.00 60.45 C ATOM 2106 CD LYS B 182 −44.810 19.791 23.894 1.00 74.61 C ATOM 2107 CE LYS B 182 −45.160 20.802 22.829 1.00 90.09 C ATOM 2108 NZ LYS B 182 −46.478 20.502 22.212 1.00 104.17 N ATOM 2109 N ALA B 183 −42.314 19.206 28.075 1.00 57.91 N ATOM 2110 CA ALA B 183 −42.348 20.175 29.186 1.00 60.15 C ATOM 2111 C ALA B 183 −40.933 20.547 29.645 1.00 63.82 C ATOM 2112 O ALA B 183 −40.628 21.728 29.711 1.00 64.74 O ATOM 2113 CB ALA B 183 −43.155 19.624 30.354 1.00 62.70 C ATOM 2114 N ASP B 184 −40.057 19.551 29.888 1.00 59.31 N ATOM 2115 CA ASP B 184 −38.658 19.805 30.266 1.00 59.12 C ATOM 2116 C ASP B 184 −37.892 20.439 29.114 1.00 59.87 C ATOM 2117 O ASP B 184 −37.064 21.317 29.358 1.00 58.39 O ATOM 2118 CB ASP B 184 −37.931 18.520 30.705 1.00 60.60 C ATOM 2119 CG ASP B 184 −38.336 17.989 32.069 1.00 75.85 C ATOM 2120 OD1 ASP B 184 −39.246 18.580 32.695 1.00 78.40 O ATOM 2121 OD2 ASP B 184 −37.744 16.980 32.511 1.00 81.02 O ATOM 2122 N TYR B 185 −38.163 20.001 27.858 1.00 54.33 N ATOM 2123 CA TYR B 185 −37.502 20.561 26.681 1.00 52.13 C ATOM 2124 C TYR B 185 −37.741 22.083 26.569 1.00 57.95 C ATOM 2125 O TYR B 185 −36.799 22.842 26.347 1.00 56.10 O ATOM 2126 CB TYR B 185 −37.919 19.823 25.390 1.00 50.32 C ATOM 2127 CG TYR B 185 −37.251 20.371 24.146 1.00 48.96 C ATOM 2128 CD1 TYR B 185 −35.883 20.192 23.937 1.00 48.51 C ATOM 2129 CD2 TYR B 185 −37.948 21.152 23.239 1.00 49.40 C ATOM 2130 CE1 TYR B 185 −35.246 20.747 22.829 1.00 45.64 C ATOM 2131 CE2 TYR B 185 −37.335 21.666 22.096 1.00 49.58 C ATOM 2132 CZ TYR B 185 −35.976 21.467 21.898 1.00 53.23 C ATOM 2133 OH TYR B 185 −35.340 21.992 20.798 1.00 51.78 O ATOM 2134 N GLU B 186 −38.984 22.526 26.783 1.00 57.91 N ATOM 2135 CA GLU B 186 −39.346 23.944 26.712 1.00 59.03 C ATOM 2136 C GLU B 186 −38.786 24.798 27.873 1.00 63.83 C ATOM 2137 O GLU B 186 −38.666 26.010 27.707 1.00 63.68 O ATOM 2138 CB GLU B 186 −40.869 24.107 26.593 1.00 61.14 C ATOM 2139 CG GLU B 186 −41.381 23.633 25.240 1.00 71.13 C ATOM 2140 CD GLU B 186 −42.880 23.453 25.094 1.00 96.57 C ATOM 2141 OE1 GLU B 186 −43.583 23.317 26.122 1.00 96.32 O ATOM 2142 OE2 GLU B 186 −43.345 23.410 23.932 1.00 90.42 O ATOM 2143 N LYS B 187 −38.405 24.181 29.014 1.00 60.66 N ATOM 2144 CA LYS B 187 −37.822 24.909 30.161 1.00 62.47 C ATOM 2145 C LYS B 187 −36.365 25.392 29.933 1.00 65.88 C ATOM 2146 O LYS B 187 −35.863 26.146 30.764 1.00 67.25 O ATOM 2147 CB LYS B 187 −37.858 24.048 31.442 1.00 65.07 C ATOM 2148 CG LYS B 187 −39.228 23.898 32.081 1.00 80.48 C ATOM 2149 CD LYS B 187 −39.160 22.926 33.265 1.00 88.59 C ATOM 2150 CE LYS B 187 −40.470 22.755 33.995 1.00 98.81 C ATOM 2151 NZ LYS B 187 −41.385 21.808 33.306 1.00 105.15 N ATOM 2152 N HIS B 188 −35.667 24.929 28.867 1.00 60.49 N ATOM 2153 CA HIS B 188 −34.268 25.306 28.622 1.00 60.34 C ATOM 2154 C HIS B 188 −34.062 25.826 27.224 1.00 61.88 C ATOM 2155 O HIS B 188 −34.881 25.566 26.341 1.00 59.68 O ATOM 2156 CB HIS B 188 −33.350 24.109 28.873 1.00 60.80 C ATOM 2157 CG HIS B 188 −33.661 23.424 30.155 1.00 66.48 C ATOM 2158 ND1 HIS B 188 −34.364 22.237 30.181 1.00 67.83 N ATOM 2159 CD2 HIS B 188 −33.429 23.822 31.423 1.00 70.78 C ATOM 2160 CE1 HIS B 188 −34.511 21.935 31.457 1.00 68.91 C ATOM 2161 NE2 HIS B 188 −33.964 22.865 32.239 1.00 71.45 N ATOM 2162 N LYS B 189 −32.942 26.536 27.020 1.00 60.07 N ATOM 2163 CA LYS B 189 −32.587 27.161 25.743 1.00 59.64 C ATOM 2164 C LYS B 189 −31.446 26.439 25.010 1.00 61.56 C ATOM 2165 O LYS B 189 −31.626 26.054 23.861 1.00 59.51 O ATOM 2166 CB LYS B 189 −32.210 28.637 25.998 1.00 63.64 C ATOM 2167 CG LYS B 189 −31.897 29.482 24.750 1.00 86.76 C ATOM 2168 CD LYS B 189 −33.007 29.508 23.672 1.00 100.09 C ATOM 2169 CE LYS B 189 −34.381 29.919 24.164 1.00 116.24 C ATOM 2170 NZ LYS B 189 −34.390 31.269 24.792 1.00 126.05 N ATOM 2171 N VAL B 190 −30.279 26.270 25.660 1.00 59.07 N ATOM 2172 CA VAL B 190 −29.086 25.696 25.022 1.00 57.13 C ATOM 2173 C VAL B 190 −29.007 24.187 25.207 1.00 59.87 C ATOM 2174 O VAL B 190 −29.004 23.721 26.339 1.00 60.57 O ATOM 2175 CB VAL B 190 −27.775 26.351 25.534 1.00 62.46 C ATOM 2176 CG1 VAL B 190 −26.594 25.966 24.641 1.00 61.26 C ATOM 2177 CG2 VAL B 190 −27.903 27.877 25.631 1.00 63.39 C ATOM 2178 N TYR B 191 −28.870 23.441 24.089 1.00 54.33 N ATOM 2179 CA TYR B 191 −28.695 21.995 24.056 1.00 52.43 C ATOM 2180 C TYR B 191 −27.375 21.755 23.359 1.00 55.56 C ATOM 2181 O TYR B 191 −27.231 22.150 22.204 1.00 53.17 O ATOM 2182 CB TYR B 191 −29.836 21.346 23.283 1.00 51.40 C ATOM 2183 CG TYR B 191 −31.107 21.388 24.087 1.00 52.89 C ATOM 2184 CD1 TYR B 191 −31.421 20.367 24.976 1.00 55.24 C ATOM 2185 CD2 TYR B 191 −31.918 22.517 24.080 1.00 52.54 C ATOM 2186 CE1 TYR B 191 −32.530 20.451 25.811 1.00 56.20 C ATOM 2187 CE2 TYR B 191 −33.045 22.603 24.892 1.00 53.78 C ATOM 2188 CZ TYR B 191 −33.343 21.567 25.764 1.00 60.22 C ATOM 2189 OH TYR B 191 −34.444 21.607 26.576 1.00 57.44 O ATOM 2190 N ALA B 192 −26.389 21.184 24.071 1.00 53.86 N ATOM 2191 CA ALA B 192 −25.047 20.981 23.534 1.00 53.95 C ATOM 2192 C ALA B 192 −24.563 19.554 23.704 1.00 58.41 C ATOM 2193 O ALA B 192 −24.882 18.900 24.688 1.00 58.37 O ATOM 2194 CB ALA B 192 −24.075 21.925 24.223 1.00 57.15 C ATOM 2195 N CYS B 193 −23.760 19.100 22.745 1.00 55.78 N ATOM 2196 CA CYS B 193 −23.131 17.791 22.738 1.00 56.08 C ATOM 2197 C CYS B 193 −21.626 18.050 22.745 1.00 57.94 C ATOM 2198 O CYS B 193 −21.149 18.719 21.839 1.00 56.41 O ATOM 2199 CB CYS B 193 −23.554 17.011 21.493 1.00 55.44 C ATOM 2200 SG CYS B 193 −22.629 15.479 21.250 1.00 59.78 S ATOM 2201 N GLU B 194 −20.890 17.564 23.755 1.00 56.17 N ATOM 2202 CA GLU B 194 −19.434 17.743 23.838 1.00 57.92 C ATOM 2203 C GLU B 194 −18.767 16.390 23.593 1.00 60.48 C ATOM 2204 O GLU B 194 −18.995 15.445 24.355 1.00 59.51 O ATOM 2205 CB GLU B 194 −19.027 18.319 25.195 1.00 61.77 C ATOM 2206 CG GLU B 194 −17.550 18.666 25.297 1.00 74.55 C ATOM 2207 CD GLU B 194 −17.129 19.093 26.686 1.00 96.82 C ATOM 2208 OE1 GLU B 194 −17.270 18.274 27.621 1.00 85.14 O ATOM 2209 OE2 GLU B 194 −16.641 20.237 26.837 1.00 99.73 O ATOM 2210 N VAL B 195 −17.935 16.313 22.536 1.00 56.28 N ATOM 2211 CA VAL B 195 −17.273 15.088 22.087 1.00 54.95 C ATOM 2212 C VAL B 195 −15.775 15.118 22.382 1.00 60.96 C ATOM 2213 O VAL B 195 −15.094 16.053 21.963 1.00 60.69 O ATOM 2214 CB VAL B 195 −17.532 14.890 20.567 1.00 56.65 C ATOM 2215 CG1 VAL B 195 −16.738 13.705 20.011 1.00 56.05 C ATOM 2216 CG2 VAL B 195 −19.027 14.741 20.283 1.00 53.80 C ATOM 2217 N THR B 196 −15.261 14.055 23.039 1.00 59.14 N ATOM 2218 CA THR B 196 −13.839 13.873 23.344 1.00 61.70 C ATOM 2219 C THR B 196 −13.334 12.670 22.527 1.00 63.94 C ATOM 2220 O THR B 196 −13.985 11.623 22.507 1.00 61.61 O ATOM 2221 CB THR B 196 −13.644 13.679 24.850 1.00 74.19 C ATOM 2222 OG1 THR B 196 −14.285 14.761 25.532 1.00 76.14 O ATOM 2223 CG2 THR B 196 −12.172 13.614 25.251 1.00 75.74 C ATOM 2224 N HIS B 197 −12.196 12.838 21.829 1.00 61.96 N ATOM 2225 CA HIS B 197 −11.605 11.795 20.982 1.00 61.16 C ATOM 2226 C HIS B 197 −10.122 12.097 20.682 1.00 68.85 C ATOM 2227 O HIS B 197 −9.753 13.262 20.519 1.00 69.73 O ATOM 2228 CB HIS B 197 −12.396 11.689 19.664 1.00 59.08 C ATOM 2229 CG HIS B 197 −11.947 10.574 18.778 1.00 61.45 C ATOM 2230 ND1 HIS B 197 −11.075 10.793 17.733 1.00 64.08 N ATOM 2231 CD2 HIS B 197 −12.277 9.264 18.806 1.00 61.56 C ATOM 2232 CE1 HIS B 197 −10.883 9.612 17.172 1.00 62.74 C ATOM 2233 NE2 HIS B 197 −11.573 8.658 17.797 1.00 61.59 N ATOM 2234 N GLN B 198 −9.292 11.039 20.580 1.00 67.35 N ATOM 2235 CA GLN B 198 −7.847 11.117 20.282 1.00 70.53 C ATOM 2236 C GLN B 198 −7.505 11.978 19.037 1.00 75.67 C ATOM 2237 O GLN B 198 −6.481 12.657 19.038 1.00 78.55 O ATOM 2238 CB GLN B 198 −7.258 9.684 20.165 1.00 72.19 C ATOM 2239 CG GLN B 198 −5.966 9.542 19.350 1.00 91.00 C ATOM 2240 CD GLN B 198 −5.167 8.296 19.647 1.00 109.12 C ATOM 2241 OE1 GLN B 198 −3.935 8.341 19.703 1.00 106.91 O ATOM 2242 NE2 GLN B 198 −5.818 7.144 19.790 1.00 99.27 N ATOM 2243 N GLY B 199 −8.365 11.973 18.008 1.00 70.22 N ATOM 2244 CA GLY B 199 −8.154 12.744 16.779 1.00 70.28 C ATOM 2245 C GLY B 199 −8.398 14.255 16.928 1.00 74.85 C ATOM 2246 O GLY B 199 −8.050 15.002 16.015 1.00 74.21 O ATOM 2247 N LEU B 200 −8.999 14.700 18.056 1.00 72.59 N ATOM 2248 CA LEU B 200 −9.288 16.108 18.346 1.00 73.59 C ATOM 2249 C LEU B 200 −8.262 16.658 19.343 1.00 82.02 C ATOM 2250 O LEU B 200 −7.944 15.975 20.320 1.00 81.96 O ATOM 2251 CB LEU B 200 −10.692 16.226 18.961 1.00 71.26 C ATOM 2252 CG LEU B 200 −11.825 15.587 18.164 1.00 72.51 C ATOM 2253 CD1 LEU B 200 −13.085 15.475 19.002 1.00 70.73 C ATOM 2254 CD2 LEU B 200 −12.068 16.347 16.875 1.00 74.05 C ATOM 2255 N SER B 201 −7.761 17.893 19.113 1.00 82.62 N ATOM 2256 CA SER B 201 −6.787 18.543 20.012 1.00 86.82 C ATOM 2257 C SER B 201 −7.428 18.852 21.369 1.00 92.27 C ATOM 2258 O SER B 201 −6.815 18.607 22.407 1.00 94.46 O ATOM 2259 CB SER B 201 −6.240 19.826 19.389 1.00 92.04 C ATOM 2260 OG SER B 201 −7.279 20.740 19.081 1.00 99.16 O ATOM 2261 N SER B 202 −8.663 19.384 21.347 1.00 87.20 N ATOM 2262 CA SER B 202 −9.465 19.681 22.538 1.00 86.89 C ATOM 2263 C SER B 202 −10.922 19.245 22.249 1.00 86.91 C ATOM 2264 O SER B 202 −11.279 19.145 21.072 1.00 84.09 O ATOM 2265 CB SER B 202 −9.408 21.172 22.873 1.00 91.46 C ATOM 2266 OG SER B 202 −9.888 21.971 21.804 1.00 97.96 O ATOM 2267 N PRO B 203 −11.758 18.993 23.292 1.00 82.94 N ATOM 2268 CA PRO B 203 −13.141 18.558 23.060 1.00 79.66 C ATOM 2269 C PRO B 203 −13.979 19.502 22.171 1.00 81.40 C ATOM 2270 O PRO B 203 −14.067 20.701 22.459 1.00 81.97 O ATOM 2271 CB PRO B 203 −13.718 18.438 24.472 1.00 82.03 C ATOM 2272 CG PRO B 203 −12.561 18.115 25.319 1.00 89.24 C ATOM 2273 CD PRO B 203 −11.369 18.792 24.704 1.00 86.84 C ATOM 2274 N VAL B 204 −14.577 18.950 21.086 1.00 74.31 N ATOM 2275 CA VAL B 204 −15.402 19.710 20.144 1.00 71.83 C ATOM 2276 C VAL B 204 −16.842 19.717 20.631 1.00 70.82 C ATOM 2277 O VAL B 204 −17.394 18.656 20.932 1.00 68.32 O ATOM 2278 CB VAL B 204 −15.288 19.152 18.703 1.00 75.22 C ATOM 2279 CG1 VAL B 204 −16.276 19.839 17.757 1.00 73.55 C ATOM 2280 CG2 VAL B 204 −13.865 19.315 18.185 1.00 77.39 C ATOM 2281 N THR B 205 −17.456 20.911 20.681 1.00 66.25 N ATOM 2282 CA THR B 205 −18.834 21.086 21.127 1.00 64.05 C ATOM 2283 C THR B 205 −19.699 21.583 19.984 1.00 65.32 C ATOM 2284 O THR B 205 −19.350 22.562 19.325 1.00 65.30 O ATOM 2285 CB THR B 205 −18.915 22.066 22.306 1.00 68.69 C ATOM 2286 OG1 THR B 205 −17.991 21.661 23.317 1.00 70.58 O ATOM 2287 CG2 THR B 205 −20.319 22.147 22.902 1.00 62.87 C ATOM 2288 N LYS B 206 −20.835 20.911 19.764 1.00 59.26 N ATOM 2289 CA LYS B 206 −21.832 21.304 18.776 1.00 57.07 C ATOM 2290 C LYS B 206 −23.101 21.561 19.579 1.00 59.72 C ATOM 2291 O LYS B 206 −23.423 20.777 20.474 1.00 58.87 O ATOM 2292 CB LYS B 206 −22.036 20.204 17.717 1.00 57.42 C ATOM 2293 CG LYS B 206 −20.795 19.932 16.858 1.00 64.65 C ATOM 2294 CD LYS B 206 −20.429 21.085 15.921 1.00 63.94 C ATOM 2295 CE LYS B 206 −19.183 20.783 15.124 1.00 68.88 C ATOM 2296 NZ LYS B 206 −19.039 21.682 13.948 1.00 79.66 N ATOM 2297 N SER B 207 −23.775 22.691 19.327 1.00 56.17 N ATOM 2298 CA SER B 207 −24.972 23.044 20.084 1.00 55.72 C ATOM 2299 C SER B 207 −25.976 23.832 19.259 1.00 58.40 C ATOM 2300 O SER B 207 −25.691 24.239 18.134 1.00 57.77 O ATOM 2301 CB SER B 207 −24.577 23.847 21.327 1.00 61.27 C ATOM 2302 OG SER B 207 −24.055 25.122 20.989 1.00 69.57 O ATOM 2303 N PHE B 208 −27.165 24.024 19.829 1.00 54.71 N ATOM 2304 CA PHE B 208 −28.222 24.838 19.236 1.00 53.49 C ATOM 2305 C PHE B 208 −29.049 25.502 20.342 1.00 58.74 C ATOM 2306 O PHE B 208 −29.086 25.008 21.477 1.00 57.67 O ATOM 2307 CB PHE B 208 −29.126 24.013 18.304 1.00 52.41 C ATOM 2308 CG PHE B 208 −29.924 22.933 18.997 1.00 52.75 C ATOM 2309 CD1 PHE B 208 −31.149 23.219 19.587 1.00 54.84 C ATOM 2310 CD2 PHE B 208 −29.460 21.627 19.044 1.00 52.67 C ATOM 2311 CE1 PHE B 208 −31.873 22.227 20.244 1.00 54.65 C ATOM 2312 CE2 PHE B 208 −30.193 20.634 19.683 1.00 54.36 C ATOM 2313 CZ PHE B 208 −31.403 20.933 20.266 1.00 52.70 C ATOM 2314 N ASN B 209 −29.718 26.609 19.993 1.00 56.87 N ATOM 2315 CA ASN B 209 −30.626 27.327 20.883 1.00 58.08 C ATOM 2316 C ASN B 209 −32.043 26.901 20.482 1.00 62.33 C ATOM 2317 O ASN B 209 −32.355 26.913 19.292 1.00 60.45 O ATOM 2318 CB ASN B 209 −30.446 28.834 20.727 1.00 58.83 C ATOM 2319 CG ASN B 209 −29.077 29.323 21.146 1.00 79.26 C ATOM 2320 OD1 ASN B 209 −28.414 28.737 22.010 1.00 68.87 O ATOM 2321 ND2 ASN B 209 −28.625 30.428 20.566 1.00 75.32 N ATOM 2322 N ARG B 210 −32.868 26.443 21.442 1.00 60.89 N ATOM 2323 CA ARG B 210 −34.233 25.986 21.149 1.00 60.59 C ATOM 2324 C ARG B 210 −35.012 27.071 20.368 1.00 69.52 C ATOM 2325 O ARG B 210 −35.045 28.226 20.789 1.00 70.27 O ATOM 2326 CB ARG B 210 −34.960 25.598 22.449 1.00 56.73 C ATOM 2327 CG ARG B 210 −36.408 25.154 22.258 1.00 56.48 C ATOM 2328 CD ARG B 210 −37.033 24.683 23.557 1.00 57.39 C ATOM 2329 NE ARG B 210 −36.967 25.712 24.593 1.00 63.13 N ATOM 2330 CZ ARG B 210 −37.759 26.782 24.679 1.00 77.20 C ATOM 2331 NH1 ARG B 210 −38.730 26.977 23.789 1.00 65.86 N ATOM 2332 NH2 ARG B 210 −37.591 27.662 25.660 1.00 59.34 N ATOM 2333 N GLY B 211 −35.593 26.698 19.214 1.00 69.48 N ATOM 2334 CA GLY B 211 −36.327 27.627 18.352 1.00 71.07 C ATOM 2335 C GLY B 211 −35.357 28.440 17.492 1.00 78.25 C ATOM 2336 O GLY B 211 −35.430 29.663 17.487 1.00 80.21 O ATOM 2337 N GLU B 212 −34.461 27.747 16.772 1.00 76.19 N ATOM 2338 CA GLU B 212 −33.428 28.309 15.888 1.00 76.94 C ATOM 2339 C GLU B 212 −32.367 29.108 16.659 1.00 81.95 C ATOM 2340 O GLU B 212 −32.603 30.233 17.092 1.00 82.60 O ATOM 2341 CB GLU B 212 −34.032 29.140 14.735 1.00 78.34 C ATOM 2342 CG GLU B 212 −35.167 28.457 13.985 1.00 91.48 C ATOM 2343 CD GLU B 212 −34.833 27.101 13.398 1.00 122.56 C ATOM 2344 OE1 GLU B 212 −34.225 27.062 12.304 1.00 124.80 O ATOM 2345 OE2 GLU B 212 −35.199 26.079 14.022 1.00 119.86 O ATOM 2346 CD CD B 9901 −18.857 6.410 6.358 1.00 126.83 CD ATOM 2347 CD CD B 9902 −33.656 22.074 34.366 1.00 101.06 CD ATOM 2348 O1 2PE B 9911 −22.872 −9.867 16.331 1.00 62.50 O ATOM 2349 C2 2PE B 9911 −22.071 −10.991 16.674 1.00 63.56 C ATOM 2350 C3 2PE B 9911 −21.935 −11.146 18.163 1.00 65.44 C ATOM 2351 O4 2PE B 9911 −20.834 −10.369 18.641 1.00 66.79 O ATOM 2352 C5 2PE B 9911 −21.040 −9.788 19.931 1.00 64.28 C ATOM 2353 C6 2PE B 9911 −19.727 −9.445 20.570 1.00 62.37 C ATOM 2354 O7 2PE B 9911 −19.494 −10.301 21.691 1.00 60.69 O ATOM 2355 C8 2PE B 9911 −18.188 −10.856 21.782 1.00 59.26 C ATOM 2356 C9 2PE B 9911 −17.274 −9.906 22.475 1.00 61.42 C ATOM 2357 O10 2PE B 9911 −15.949 −10.431 22.475 1.00 63.12 O ATOM 2358 C11 2PE B 9911 −14.979 −9.603 21.849 1.00 65.29 C ATOM 2359 C12 2PE B 9911 −14.612 −8.454 22.735 1.00 69.11 C ATOM 2360 O13 2PE B 9911 −14.469 −7.280 21.939 1.00 72.89 O ATOM 2361 C14 2PE B 9911 −14.725 −6.052 22.615 1.00 77.60 C ATOM 2362 C15 2PE B 9911 −15.447 −5.108 21.699 1.00 80.79 C ATOM 2363 O16 2PE B 9911 −16.827 −5.016 22.050 1.00 84.78 O ATOM 2364 C17 2PE B 9911 −17.094 −4.168 23.171 1.00 90.27 C ATOM 2365 C18 2PE B 9911 −18.054 −4.834 24.111 1.00 91.97 C ATOM 2366 O19 2PE B 9911 −17.687 −4.558 25.466 1.00 95.36 O ATOM 2367 C20 2PE B 9911 −17.491 −5.710 26.284 1.00 96.33 C ATOM 2368 C21 2PE B 9911 −18.803 −6.194 26.819 1.00 95.30 C ATOM 2369 O22 2PE B 9911 −18.660 −7.493 27.385 1.00 94.55 O ATOM 2370 C23 2PE B 9911 −19.090 −8.542 26.525 1.00 91.87 C ATOM 2371 C24 2PE B 9911 −18.693 −9.866 27.094 1.00 92.15 C ATOM 2372 O25 2PE B 9911 −19.003 −10.901 26.162 1.00 89.40 O ATOM 2373 C26 2PE B 9911 −18.469 −12.180 26.492 1.00 87.56 C ATOM 2374 C27 2PE B 9911 −19.251 −12.799 27.621 1.00 86.27 C ATOM 2375 028 2PE B 9911 −19.209 −14.218 27.573 1.00 84.51 O ATOM 2376 N GLU C 1 −22.618 −29.854 1.438 1.00 63.58 N ATOM 2377 CA GLU C 1 −23.717 −30.821 1.519 1.00 63.31 C ATOM 2378 C GLU C 1 −24.332 −30.855 2.927 1.00 61.30 C ATOM 2379 O GLU C 1 −25.562 −30.934 3.036 1.00 61.09 O ATOM 2380 CB GLU C 1 −23.250 −32.227 1.101 1.00 66.84 C ATOM 2381 CG GLU C 1 −24.314 −33.319 1.157 1.00 82.05 C ATOM 2382 CD GLU C 1 −25.611 −33.021 0.422 1.00 109.67 C ATOM 2383 OE1 GLU C 1 −25.551 −32.432 −0.682 1.00 110.68 O ATOM 2384 OE2 GLU C 1 −26.689 −33.395 0.940 1.00 104.39 O ATOM 2385 N ILE C 2 −23.504 −30.833 3.999 1.00 51.48 N ATOM 2386 CA ILE C 2 −24.059 −30.861 5.352 1.00 47.04 C ATOM 2387 C ILE C 2 −24.625 −29.480 5.729 1.00 47.71 C ATOM 2388 O ILE C 2 −23.925 −28.470 5.624 1.00 45.78 O ATOM 2389 CB ILE C 2 −23.058 −31.378 6.415 1.00 48.86 C ATOM 2390 CG1 ILE C 2 −22.700 −32.866 6.161 1.00 49.65 C ATOM 2391 CG2 ILE C 2 −23.644 −31.168 7.838 1.00 46.14 C ATOM 2392 CD1 ILE C 2 −21.523 −33.450 7.026 1.00 47.83 C ATOM 2393 N GLN C 3 −25.870 −29.453 6.222 1.00 43.58 N ATOM 2394 CA GLN C 3 −26.493 −28.215 6.675 1.00 42.22 C ATOM 2395 C GLN C 3 −27.422 −28.456 7.873 1.00 43.24 C ATOM 2396 O GLN C 3 −28.128 −29.463 7.913 1.00 42.56 O ATOM 2397 CB GLN C 3 −27.279 −27.571 5.520 1.00 44.23 C ATOM 2398 CG GLN C 3 −27.799 −26.166 5.849 1.00 66.49 C ATOM 2399 CD GLN C 3 −27.461 −25.147 4.789 1.00 94.56 C ATOM 2400 OE1 GLN C 3 −26.284 −24.843 4.550 1.00 93.02 O ATOM 2401 NE2 GLN C 3 −28.476 −24.556 4.160 1.00 84.87 N ATOM 2402 N LEU C 4 −27.416 −27.515 8.839 1.00 37.54 N ATOM 2403 CA LEU C 4 −28.322 −27.497 9.987 1.00 35.69 C ATOM 2404 C LEU C 4 −29.240 −26.293 9.752 1.00 37.93 C ATOM 2405 O LEU C 4 −28.736 −25.183 9.626 1.00 34.85 O ATOM 2406 CB LEU C 4 −27.554 −27.318 11.308 1.00 34.43 C ATOM 2407 CG LEU C 4 −26.555 −28.411 11.693 1.00 39.46 C ATOM 2408 CD1 LEU C 4 −26.104 −28.237 13.129 1.00 37.91 C ATOM 2409 CD2 LEU C 4 −27.122 −29.796 11.492 1.00 41.20 C ATOM 2410 N VAL C 5 −30.561 −26.514 9.621 1.00 35.79 N ATOM 2411 CA VAL C 5 −31.519 −25.437 9.367 1.00 35.25 C ATOM 2412 C VAL C 5 −32.391 −25.265 10.586 1.00 35.99 C ATOM 2413 O VAL C 5 −33.154 −26.161 10.918 1.00 34.91 O ATOM 2414 CB VAL C 5 −32.386 −25.701 8.102 1.00 40.70 C ATOM 2415 CG1 VAL C 5 −33.389 −24.564 7.876 1.00 40.07 C ATOM 2416 CG2 VAL C 5 −31.506 −25.886 6.869 1.00 41.08 C ATOM 2417 N GLN C 6 −32.333 −24.097 11.211 1.00 33.06 N ATOM 2418 CA GLN C 6 −33.158 −23.799 12.378 1.00 32.21 C ATOM 2419 C GLN C 6 −34.440 −23.054 12.027 1.00 36.16 C ATOM 2420 O GLN C 6 −34.502 −22.364 11.005 1.00 34.87 O ATOM 2421 CB GLN C 6 −32.361 −22.988 13.389 1.00 31.67 C ATOM 2422 CG GLN C 6 −31.222 −23.775 13.990 1.00 33.35 C ATOM 2423 CD GLN C 6 −30.507 −22.998 15.048 1.00 37.99 C ATOM 2424 OE1 GLN C 6 −29.304 −22.789 14.961 1.00 32.51 O ATOM 2425 NE2 GLN C 6 −31.204 −22.608 16.108 1.00 34.83 N ATOM 2426 N SER C 7 −35.442 −23.145 12.912 1.00 33.10 N ATOM 2427 CA SER C 7 −36.714 −22.436 12.728 1.00 32.33 C ATOM 2428 C SER C 7 −36.530 −20.904 12.916 1.00 34.33 C ATOM 2429 O SER C 7 −35.486 −20.441 13.402 1.00 32.67 O ATOM 2430 CB SER C 7 −37.790 −22.996 13.663 1.00 34.45 C ATOM 2431 OG SER C 7 −37.340 −23.094 15.005 1.00 35.38 O ATOM 2432 N GLY C 8 −37.536 −20.129 12.496 1.00 31.79 N ATOM 2433 CA GLY C 8 −37.499 −18.664 12.499 1.00 30.41 C ATOM 2434 C GLY C 8 −37.561 −17.981 13.867 1.00 36.01 C ATOM 2435 O GLY C 8 −37.760 −18.623 14.905 1.00 34.46 O ATOM 2436 N ALA C 9 −37.403 −16.640 13.835 1.00 33.40 N ATOM 2437 CA ALA C 9 −37.415 −15.754 15.005 1.00 32.51 C ATOM 2438 C ALA C 9 −38.680 −15.933 15.864 1.00 36.53 C ATOM 2439 O ALA C 9 −39.776 −16.123 15.326 1.00 35.95 O ATOM 2440 CB ALA C 9 −37.297 −14.299 14.553 1.00 32.54 C ATOM 2441 N GLU C 10 −38.514 −15.861 17.196 1.00 32.54 N ATOM 2442 CA GLU C 10 −39.591 −16.043 18.164 1.00 33.03 C ATOM 2443 C GLU C 10 −39.707 −14.842 19.084 1.00 37.36 C ATOM 2444 O GLU C 10 −38.696 −14.324 19.553 1.00 36.85 O ATOM 2445 CB GLU C 10 −39.315 −17.292 19.032 1.00 34.31 C ATOM 2446 CG GLU C 10 −39.313 −18.599 18.253 1.00 39.42 C ATOM 2447 CD GLU C 10 −40.668 −19.160 17.863 1.00 50.94 C ATOM 2448 OE1 GLU C 10 −41.708 −18.572 18.239 1.00 42.37 O ATOM 2449 OE2 GLU C 10 −40.684 −20.185 17.146 1.00 50.90 O ATOM 2450 N VAL C 11 −40.946 −14.421 19.373 1.00 35.06 N ATOM 2451 CA VAL C 11 −41.231 −13.301 20.272 1.00 34.90 C ATOM 2452 C VAL C 11 −42.306 −13.780 21.230 1.00 40.99 C ATOM 2453 O VAL C 11 −43.376 −14.192 20.788 1.00 42.78 O ATOM 2454 CB VAL C 11 −41.638 −12.035 19.496 1.00 36.50 C ATOM 2455 CG1 VAL C 11 −41.698 −10.828 20.427 1.00 35.68 C ATOM 2456 CG2 VAL C 11 −40.656 −11.786 18.359 1.00 35.29 C ATOM 2457 N LYS C 12 −41.998 −13.797 22.526 1.00 38.39 N ATOM 2458 CA LYS C 12 −42.875 −14.357 23.553 1.00 39.13 C ATOM 2459 C LYS C 12 −43.030 −13.439 24.750 1.00 43.69 C ATOM 2460 O LYS C 12 −42.196 −12.567 24.985 1.00 41.69 O ATOM 2461 CB LYS C 12 −42.269 −15.685 24.050 1.00 40.54 C ATOM 2462 CG LYS C 12 −42.082 −16.767 22.986 1.00 40.87 C ATOM 2463 CD LYS C 12 −43.385 −17.464 22.642 1.00 43.40 C ATOM 2464 CE LYS C 12 −43.234 −18.398 21.470 1.00 49.33 C ATOM 2465 NZ LYS C 12 −44.526 −19.027 21.098 1.00 60.76 N ATOM 2466 N LYS C 13 −44.084 −13.682 25.538 1.00 43.27 N ATOM 2467 CA LYS C 13 −44.349 −12.946 26.772 1.00 44.58 C ATOM 2468 C LYS C 13 −43.672 −13.730 27.900 1.00 48.51 C ATOM 2469 O LYS C 13 −43.477 −14.941 27.741 1.00 47.08 O ATOM 2470 CB LYS C 13 −45.860 −12.867 27.055 1.00 48.66 C ATOM 2471 CG LYS C 13 −46.686 −12.183 25.969 1.00 54.31 C ATOM 2472 CD LYS C 13 −46.569 −10.668 25.985 1.00 57.75 C ATOM 2473 CE LYS C 13 −47.609 −10.047 25.081 1.00 65.70 C ATOM 2474 NZ LYS C 13 −47.421 −8.585 24.928 1.00 75.98 N ATOM 2475 N PRO C 14 −43.359 −13.079 29.052 1.00 46.94 N ATOM 2476 CA PRO C 14 −42.788 −13.806 30.180 1.00 47.12 C ATOM 2477 C PRO C 14 −43.771 −14.860 30.685 1.00 51.78 C ATOM 2478 O PRO C 14 −44.981 −14.614 30.686 1.00 51.43 O ATOM 2479 CB PRO C 14 −42.535 −12.708 31.217 1.00 49.87 C ATOM 2480 CG PRO C 14 −42.284 −11.487 30.418 1.00 52.81 C ATOM 2481 CD PRO C 14 −42.928 −11.667 29.071 1.00 47.65 C ATOM 2482 N GLY C 15 −43.257 −16.053 31.042 1.00 48.78 N ATOM 2483 CA GLY C 15 −44.082 −17.158 31.533 1.00 50.33 C ATOM 2484 C GLY C 15 −44.502 −18.130 30.431 1.00 53.71 C ATOM 2485 O GLY C 15 −44.949 −19.224 30.750 1.00 54.84 O ATOM 2486 N ALA C 16 −44.368 −17.752 29.141 1.00 49.31 N ATOM 2487 CA ALA C 16 −44.747 −18.631 28.027 1.00 48.16 C ATOM 2488 C ALA C 16 −43.645 −19.659 27.746 1.00 49.44 C ATOM 2489 O ALA C 16 −42.606 −19.656 28.407 1.00 47.16 O ATOM 2490 CB ALA C 16 −45.003 −17.792 26.780 1.00 47.36 C ATOM 2491 N SER C 17 −43.867 −20.529 26.757 1.00 46.84 N ATOM 2492 CA SER C 17 −42.863 −21.495 26.317 1.00 45.71 C ATOM 2493 C SER C 17 −42.603 −21.337 24.814 1.00 48.77 C ATOM 2494 O SER C 17 −43.441 −20.803 24.076 1.00 47.69 O ATOM 2495 CB SER C 17 −43.270 −22.926 26.675 1.00 51.76 C ATOM 2496 OG SER C 17 −44.464 −23.326 26.024 1.00 66.12 O ATOM 2497 N VAL C 18 −41.405 −21.746 24.378 1.00 44.66 N ATOM 2498 CA VAL C 18 −40.986 −21.671 22.971 1.00 42.57 C ATOM 2499 C VAL C 18 −40.295 −22.984 22.619 1.00 45.17 C ATOM 2500 O VAL C 18 −39.628 −23.557 23.468 1.00 44.37 O ATOM 2501 CB VAL C 18 −40.069 −20.431 22.706 1.00 44.23 C ATOM 2502 CG1 VAL C 18 −38.754 −20.510 23.478 1.00 43.39 C ATOM 2503 CG2 VAL C 18 −39.800 −20.240 21.221 1.00 42.49 C ATOM 2504 N LYS C 19 −40.449 −23.445 21.376 1.00 42.15 N ATOM 2505 CA LYS C 19 −39.828 −24.671 20.886 1.00 41.47 C ATOM 2506 C LYS C 19 −39.072 −24.335 19.609 1.00 42.91 C ATOM 2507 O LYS C 19 −39.684 −23.911 18.631 1.00 43.64 O ATOM 2508 CB LYS C 19 −40.888 −25.751 20.634 1.00 45.01 C ATOM 2509 CG LYS C 19 −40.308 −27.156 20.448 1.00 49.05 C ATOM 2510 CD LYS C 19 −41.418 −28.215 20.426 1.00 60.62 C ATOM 2511 CE LYS C 19 −40.902 −29.617 20.650 1.00 79.35 C ATOM 2512 NZ LYS C 19 −42.009 −30.605 20.812 1.00 88.79 N ATOM 2513 N VAL C 20 −37.738 −24.469 19.637 1.00 37.38 N ATOM 2514 CA VAL C 20 −36.871 −24.175 18.489 1.00 35.44 C ATOM 2515 C VAL C 20 −36.495 −25.507 17.860 1.00 38.00 C ATOM 2516 O VAL C 20 −36.146 −26.430 18.585 1.00 36.91 O ATOM 2517 CB VAL C 20 −35.611 −23.375 18.931 1.00 37.50 C ATOM 2518 CG1 VAL C 20 −34.650 −23.152 17.759 1.00 36.60 C ATOM 2519 CG2 VAL C 20 −36.016 −22.038 19.552 1.00 36.75 C ATOM 2520 N SER C 21 −36.574 −25.615 16.531 1.00 34.47 N ATOM 2521 CA SER C 21 −36.224 −26.848 15.830 1.00 34.87 C ATOM 2522 C SER C 21 −34.903 −26.650 15.077 1.00 40.61 C ATOM 2523 O SER C 21 −34.486 −25.518 14.820 1.00 39.50 O ATOM 2524 CB SER C 21 −37.344 −27.262 14.883 1.00 37.26 C ATOM 2525 OG SER C 21 −37.457 −26.351 13.803 1.00 48.80 O ATOM 2526 N CYS C 22 −34.236 −27.757 14.773 1.00 37.95 N ATOM 2527 CA CYS C 22 −32.951 −27.774 14.074 1.00 38.24 C ATOM 2528 C CYS C 22 −32.951 −29.009 13.165 1.00 42.29 C ATOM 2529 O CYS C 22 −32.783 −30.117 13.660 1.00 43.00 O ATOM 2530 CB CYS C 22 −31.816 −27.820 15.100 1.00 38.86 C ATOM 2531 SG CYS C 22 −30.172 −28.148 14.408 1.00 42.75 S ATOM 2532 N LYS C 23 −33.185 −28.822 11.862 1.00 39.28 N ATOM 2533 CA LYS C 23 −33.231 −29.916 10.893 1.00 39.83 C ATOM 2534 C LYS C 23 −31.852 −30.134 10.289 1.00 43.79 C ATOM 2535 O LYS C 23 −31.302 −29.223 9.686 1.00 42.31 O ATOM 2536 CB LYS C 23 −34.254 −29.611 9.787 1.00 43.14 C ATOM 2537 CG LYS C 23 −34.474 −30.785 8.825 1.00 52.94 C ATOM 2538 CD LYS C 23 −35.742 −30.618 7.995 1.00 58.08 C ATOM 2539 CE LYS C 23 −35.928 −31.725 6.979 1.00 60.42 C ATOM 2540 NZ LYS C 23 −36.619 −32.901 7.555 1.00 59.48 N ATOM 2541 N ALA C 24 −31.310 −31.351 10.425 1.00 42.99 N ATOM 2542 CA ALA C 24 −29.998 −31.718 9.891 1.00 43.47 C ATOM 2543 C ALA C 24 −30.144 −32.476 8.573 1.00 48.66 C ATOM 2544 O ALA C 24 −31.098 −33.241 8.411 1.00 49.47 O ATOM 2545 CB ALA C 24 −29.276 −32.604 10.890 1.00 44.42 C ATOM 2546 N SER C 25 −29.178 −32.302 7.652 1.00 44.71 N ATOM 2547 CA SER C 25 −29.148 −33.037 6.384 1.00 45.72 C ATOM 2548 C SER C 25 −27.706 −33.238 5.922 1.00 50.57 C ATOM 2549 O SER C 25 −26.822 −32.512 6.371 1.00 49.51 O ATOM 2550 CB SER C 25 −29.960 −32.303 5.314 1.00 49.16 C ATOM 2551 OG SER C 25 −29.436 −31.022 5.006 1.00 50.85 O ATOM 2552 N GLY C 26 −27.468 −34.242 5.053 1.00 49.05 N ATOM 2553 CA GLY C 26 −26.155 −34.522 4.467 1.00 49.32 C ATOM 2554 C GLY C 26 −25.287 −35.531 5.217 1.00 53.45 C ATOM 2555 O GLY C 26 −24.162 −35.785 4.780 1.00 54.80 O ATOM 2556 N TYR C 27 −25.778 −36.114 6.324 1.00 48.38 N ATOM 2557 CA TYR C 27 −25.002 −37.088 7.103 1.00 46.86 C ATOM 2558 C TYR C 27 −25.932 −37.981 7.925 1.00 51.13 C ATOM 2559 O TYR C 27 −27.143 −37.750 7.938 1.00 50.12 O ATOM 2560 CB TYR C 27 −24.001 −36.349 8.015 1.00 45.84 C ATOM 2561 CG TYR C 27 −24.643 −35.614 9.175 1.00 43.92 C ATOM 2562 CD1 TYR C 27 −25.131 −34.323 9.024 1.00 44.44 C ATOM 2563 CD2 TYR C 27 −24.730 −36.201 10.432 1.00 43.61 C ATOM 2564 CE1 TYR C 27 −25.684 −33.627 10.100 1.00 43.30 C ATOM 2565 CE2 TYR C 27 −25.284 −35.520 11.515 1.00 42.32 C ATOM 2566 CZ TYR C 27 −25.766 −34.234 11.345 1.00 47.43 C ATOM 2567 OH TYR C 27 −26.322 −33.590 12.424 1.00 44.79 O ATOM 2568 N THR C 28 −25.363 −38.986 8.621 1.00 49.09 N ATOM 2569 CA THR C 28 −26.132 −39.907 9.466 1.00 49.25 C ATOM 2570 C THR C 28 −26.422 −39.195 10.792 1.00 51.29 C ATOM 2571 O THR C 28 −25.548 −39.106 11.649 1.00 50.53 O ATOM 2572 CB THR C 28 −25.378 −41.240 9.638 1.00 59.68 C ATOM 2573 OG1 THR C 28 −25.036 −41.741 8.344 1.00 59.87 O ATOM 2574 CG2 THR C 28 −26.197 −42.289 10.394 1.00 58.03 C ATOM 2575 N PHE C 29 −27.644 −38.652 10.926 1.00 46.66 N ATOM 2576 CA PHE C 29 −28.109 −37.880 12.083 1.00 44.45 C ATOM 2577 C PHE C 29 −27.841 −38.536 13.439 1.00 46.44 C ATOM 2578 O PHE C 29 −27.371 −37.866 14.353 1.00 44.51 O ATOM 2579 CB PHE C 29 −29.618 −37.587 11.943 1.00 45.95 C ATOM 2580 CG PHE C 29 −30.193 −36.720 13.040 1.00 45.40 C ATOM 2581 CD1 PHE C 29 −29.801 −35.396 13.178 1.00 45.67 C ATOM 2582 CD2 PHE C 29 −31.106 −37.239 13.954 1.00 46.43 C ATOM 2583 CE1 PHE C 29 −30.325 −34.596 14.194 1.00 45.11 C ATOM 2584 CE2 PHE C 29 −31.652 −36.430 14.951 1.00 47.75 C ATOM 2585 CZ PHE C 29 −31.255 −35.117 15.069 1.00 44.49 C ATOM 2586 N THR C 30 −28.108 −39.844 13.546 1.00 44.66 N ATOM 2587 CA THR C 30 −27.971 −40.605 14.793 1.00 44.62 C ATOM 2588 C THR C 30 −26.521 −40.998 15.170 1.00 46.75 C ATOM 2589 O THR C 30 −26.341 −41.611 16.214 1.00 44.59 O ATOM 2590 CB THR C 30 −28.879 −41.843 14.734 1.00 54.91 C ATOM 2591 OG1 THR C 30 −28.458 −42.673 13.653 1.00 58.28 O ATOM 2592 CG2 THR C 30 −30.354 −41.475 14.559 1.00 52.29 C ATOM 2593 N ASN C 31 −25.495 −40.624 14.366 1.00 43.46 N ATOM 2594 CA ASN C 31 −24.084 −40.936 14.667 1.00 43.21 C ATOM 2595 C ASN C 31 −23.329 −39.787 15.353 1.00 44.04 C ATOM 2596 O ASN C 31 −22.212 −39.999 15.817 1.00 44.02 O ATOM 2597 CB ASN C 31 −23.344 −41.333 13.378 1.00 43.58 C ATOM 2598 CG ASN C 31 −23.662 −42.722 12.874 1.00 59.56 C ATOM 2599 OD1 ASN C 31 −24.315 −43.539 13.539 1.00 52.70 O ATOM 2600 ND2 ASN C 31 −23.206 −43.018 11.674 1.00 53.16 N ATOM 2601 N TYR C 32 −23.923 −38.584 15.414 1.00 40.04 N ATOM 2602 CA TYR C 32 −23.294 −37.394 15.983 1.00 38.44 C ATOM 2603 C TYR C 32 −24.233 −36.723 16.960 1.00 41.02 C ATOM 2604 O TYR C 32 −25.401 −36.561 16.637 1.00 40.60 O ATOM 2605 CB TYR C 32 −22.961 −36.394 14.850 1.00 38.37 C ATOM 2606 CG TYR C 32 −22.034 −36.960 13.799 1.00 40.01 C ATOM 2607 CD1 TYR C 32 −22.526 −37.722 12.745 1.00 41.48 C ATOM 2608 CD2 TYR C 32 −20.662 −36.775 13.883 1.00 41.23 C ATOM 2609 CE1 TYR C 32 −21.673 −38.280 11.796 1.00 41.39 C ATOM 2610 CE2 TYR C 32 −19.796 −37.339 12.949 1.00 43.02 C ATOM 2611 CZ TYR C 32 −20.306 −38.083 11.898 1.00 48.67 C ATOM 2612 OH TYR C 32 −19.468 −38.606 10.938 1.00 49.51 O ATOM 2613 N GLY C 33 −23.733 −36.290 18.125 1.00 37.23 N ATOM 2614 CA GLY C 33 −24.545 −35.563 19.098 1.00 36.84 C ATOM 2615 C GLY C 33 −24.838 −34.150 18.599 1.00 40.03 C ATOM 2616 O GLY C 33 −24.157 −33.661 17.693 1.00 39.15 O ATOM 2617 N MET C 34 −25.861 −33.501 19.173 1.00 35.88 N ATOM 2618 CA MET C 34 −26.236 −32.144 18.797 1.00 34.40 C ATOM 2619 C MET C 34 −26.134 −31.239 20.019 1.00 39.38 C ATOM 2620 O MET C 34 −26.831 −31.463 21.009 1.00 40.01 O ATOM 2621 CB MET C 34 −27.666 −32.111 18.229 1.00 35.96 C ATOM 2622 CG MET C 34 −28.039 −30.776 17.579 1.00 37.38 C ATOM 2623 SD MET C 34 −26.998 −30.373 16.152 1.00 39.09 S ATOM 2624 CE MET C 34 −27.629 −31.496 14.976 1.00 35.89 C ATOM 2625 N ASN C 35 −25.253 −30.232 19.949 1.00 35.27 N ATOM 2626 CA ASN C 35 −25.070 −29.236 21.010 1.00 34.40 C ATOM 2627 C ASN C 35 −26.072 −28.130 20.849 1.00 37.48 C ATOM 2628 O ASN C 35 −26.507 −27.859 19.735 1.00 36.13 O ATOM 2629 CB ASN C 35 −23.682 −28.578 20.927 1.00 32.88 C ATOM 2630 CG ASN C 35 −22.531 −29.545 21.095 1.00 45.03 C ATOM 2631 OD1 ASN C 35 −22.213 −29.971 22.211 1.00 36.36 O ATOM 2632 ND2 ASN C 35 −21.893 −29.927 20.006 1.00 37.11 N ATOM 2633 N TRP C 36 −26.393 −27.451 21.956 1.00 34.97 N ATOM 2634 CA TRP C 36 −27.235 −26.258 21.970 1.00 33.48 C ATOM 2635 C TRP C 36 −26.364 −25.194 22.617 1.00 35.71 C ATOM 2636 O TRP C 36 −25.736 −25.477 23.633 1.00 35.70 O ATOM 2637 CB TRP C 36 −28.554 −26.471 22.725 1.00 32.77 C ATOM 2638 CG TRP C 36 −29.505 −27.349 21.972 1.00 34.47 C ATOM 2639 CD1 TRP C 36 −29.633 −28.704 22.096 1.00 38.31 C ATOM 2640 CD2 TRP C 36 −30.277 −26.977 20.822 1.00 33.76 C ATOM 2641 NE1 TRP C 36 −30.519 −29.182 21.154 1.00 37.71 N ATOM 2642 CE2 TRP C 36 −30.908 −28.147 20.343 1.00 37.56 C ATOM 2643 CE3 TRP C 36 −30.552 −25.753 20.188 1.00 34.01 C ATOM 2644 CZ2 TRP C 36 −31.801 −28.127 19.270 1.00 36.52 C ATOM 2645 CZ3 TRP C 36 −31.473 −25.728 19.152 1.00 34.99 C ATOM 2646 CH2 TRP C 36 −32.075 −26.905 18.693 1.00 36.19 C ATOM 2647 N VAL C 37 −26.225 −24.029 21.962 1.00 31.77 N ATOM 2648 CA VAL C 37 −25.368 −22.931 22.408 1.00 31.68 C ATOM 2649 C VAL C 37 −26.214 −21.647 22.471 1.00 35.29 C ATOM 2650 O VAL C 37 −26.931 −21.340 21.522 1.00 33.99 O ATOM 2651 CB VAL C 37 −24.172 −22.788 21.412 1.00 34.93 C ATOM 2652 CG1 VAL C 37 −23.336 −21.531 21.676 1.00 34.09 C ATOM 2653 CG2 VAL C 37 −23.300 −24.046 21.423 1.00 35.24 C ATOM 2654 N LYS C 38 −26.111 −20.898 23.574 1.00 33.55 N ATOM 2655 CA LYS C 38 −26.828 −19.631 23.766 1.00 32.90 C ATOM 2656 C LYS C 38 −25.879 −18.433 23.539 1.00 36.90 C ATOM 2657 O LYS C 38 −24.732 −18.444 24.001 1.00 35.60 O ATOM 2658 CB LYS C 38 −27.403 −19.567 25.201 1.00 34.99 C ATOM 2659 CG LYS C 38 −28.113 −18.248 25.543 1.00 35.35 C ATOM 2660 CD LYS C 38 −28.588 −18.249 26.978 1.00 39.90 C ATOM 2661 CE LYS C 38 −29.184 −16.929 27.403 1.00 34.43 C ATOM 2662 NZ LYS C 38 −29.763 −17.022 28.775 1.00 38.69 N ATOM 2663 N GLN C 39 −26.384 −17.382 22.865 1.00 33.51 N ATOM 2664 CA GLN C 39 −25.650 −16.136 22.683 1.00 32.91 C ATOM 2665 C GLN C 39 −26.573 −14.967 23.027 1.00 36.70 C ATOM 2666 O GLN C 39 −27.387 −14.546 22.200 1.00 35.72 O ATOM 2667 CB GLN C 39 −25.074 −16.007 21.265 1.00 33.64 C ATOM 2668 CG GLN C 39 −24.267 −14.717 21.087 1.00 35.85 C ATOM 2669 CD GLN C 39 −23.282 −14.813 19.955 1.00 40.10 C ATOM 2670 OE1 GLN C 39 −23.590 −15.333 18.884 1.00 34.17 O ATOM 2671 NE2 GLN C 39 −22.099 −14.253 20.121 1.00 33.15 N ATOM 2672 N ALA C 40 −26.462 −14.465 24.262 1.00 35.19 N ATOM 2673 CA ALA C 40 −27.256 −13.328 24.726 1.00 35.91 C ATOM 2674 C ALA C 40 −26.802 −12.042 23.985 1.00 42.15 C ATOM 2675 O ALA C 40 −25.671 −12.015 23.489 1.00 41.64 O ATOM 2676 CB ALA C 40 −27.120 −13.172 26.237 1.00 37.19 C ATOM 2677 N PRO C 41 −27.681 −11.015 23.846 1.00 40.24 N ATOM 2678 CA PRO C 41 −27.300 −9.803 23.094 1.00 40.39 C ATOM 2679 C PRO C 41 −26.025 −9.141 23.636 1.00 44.02 C ATOM 2680 O PRO C 41 −25.911 −8.909 24.837 1.00 44.30 O ATOM 2681 CB PRO C 41 −28.538 −8.894 23.208 1.00 42.20 C ATOM 2682 CG PRO C 41 −29.652 −9.805 23.576 1.00 46.06 C ATOM 2683 CD PRO C 41 −29.025 −10.859 24.436 1.00 41.75 C ATOM 2684 N GLY C 42 −25.036 −8.940 22.757 1.00 40.30 N ATOM 2685 CA GLY C 42 −23.757 −8.345 23.127 1.00 40.77 C ATOM 2686 C GLY C 42 −22.812 −9.293 23.876 1.00 45.52 C ATOM 2687 O GLY C 42 −21.745 −8.838 24.279 1.00 48.05 O ATOM 2688 N GLN C 43 −23.160 −10.596 24.043 1.00 39.62 N ATOM 2689 CA GLN C 43 −22.328 −11.545 24.791 1.00 40.29 C ATOM 2690 C GLN C 43 −21.691 −12.601 23.865 1.00 43.71 C ATOM 2691 O GLN C 43 −21.931 −12.611 22.655 1.00 41.68 O ATOM 2692 CB GLN C 43 −23.168 −12.233 25.898 1.00 42.01 C ATOM 2693 CG GLN C 43 −23.863 −11.283 26.883 1.00 58.06 C ATOM 2694 CD GLN C 43 −22.892 −10.511 27.738 1.00 86.83 C ATOM 2695 OE1 GLN C 43 −22.255 −11.069 28.638 1.00 88.11 O ATOM 2696 NE2 GLN C 43 −22.754 −9.211 27.490 1.00 80.98 N ATOM 2697 N GLY C 44 −20.879 −13.487 24.455 1.00 42.03 N ATOM 2698 CA GLY C 44 −20.186 −14.559 23.752 1.00 41.97 C ATOM 2699 C GLY C 44 −21.034 −15.828 23.709 1.00 44.75 C ATOM 2700 O GLY C 44 −22.256 −15.760 23.819 1.00 44.03 O ATOM 2701 N LEU C 45 −20.378 −16.986 23.571 1.00 41.38 N ATOM 2702 CA LEU C 45 −21.051 −18.286 23.471 1.00 38.74 C ATOM 2703 C LEU C 45 −21.096 −19.009 24.820 1.00 41.07 C ATOM 2704 O LEU C 45 −20.115 −19.017 25.558 1.00 41.06 O ATOM 2705 CB LEU C 45 −20.354 −19.142 22.400 1.00 37.85 C ATOM 2706 CG LEU C 45 −20.165 −18.444 21.029 1.00 40.26 C ATOM 2707 CD1 LEU C 45 −19.302 −19.273 20.106 1.00 39.97 C ATOM 2708 CD2 LEU C 45 −21.512 −18.127 20.365 1.00 39.04 C ATOM 2709 N LYS C 46 −22.254 −19.604 25.137 1.00 36.64 N ATOM 2710 CA LYS C 46 −22.491 −20.356 26.363 1.00 36.49 C ATOM 2711 C LYS C 46 −23.068 −21.720 25.970 1.00 37.74 C ATOM 2712 O LYS C 46 −24.114 −21.778 25.336 1.00 35.53 O ATOM 2713 CB LYS C 46 −23.482 −19.579 27.259 1.00 38.67 C ATOM 2714 CG LYS C 46 −23.730 −20.181 28.639 1.00 47.96 C ATOM 2715 CD LYS C 46 −22.544 −20.014 29.567 1.00 59.13 C ATOM 2716 CE LYS C 46 −22.893 −20.303 31.011 1.00 66.31 C ATOM 2717 NZ LYS C 46 −21.727 −20.086 31.910 1.00 72.38 N ATOM 2718 N TRP C 47 −22.391 −22.810 26.350 1.00 36.18 N ATOM 2719 CA TRP C 47 −22.844 −24.174 26.054 1.00 35.61 C ATOM 2720 C TRP C 47 −24.004 −24.522 26.986 1.00 39.00 C ATOM 2721 O TRP C 47 −23.853 −24.431 28.197 1.00 39.05 O ATOM 2722 CB TRP C 47 −21.693 −25.160 26.260 1.00 34.55 C ATOM 2723 CG TRP C 47 −21.961 −26.568 25.810 1.00 35.04 C ATOM 2724 CD1 TRP C 47 −21.904 −27.043 24.532 1.00 37.01 C ATOM 2725 CD2 TRP C 47 −22.128 −27.714 26.658 1.00 35.28 C ATOM 2726 NE1 TRP C 47 −22.099 −28.405 24.526 1.00 36.66 N ATOM 2727 CE2 TRP C 47 −22.197 −28.848 25.820 1.00 38.90 C ATOM 2728 CE3 TRP C 47 −22.225 −27.893 28.048 1.00 37.36 C ATOM 2729 CZ2 TRP C 47 −22.360 −30.139 26.322 1.00 38.56 C ATOM 2730 CZ3 TRP C 47 −22.374 −29.180 28.548 1.00 39.47 C ATOM 2731 CH2 TRP C 47 −22.451 −30.283 27.690 1.00 39.81 C ATOM 2732 N MET C 48 −25.164 −24.876 26.420 1.00 34.50 N ATOM 2733 CA MET C 48 −26.359 −25.221 27.204 1.00 34.00 C ATOM 2734 C MET C 48 −26.370 −26.703 27.577 1.00 38.45 C ATOM 2735 O MET C 48 −26.899 −27.080 28.623 1.00 38.74 O ATOM 2736 CB MET C 48 −27.624 −24.887 26.406 1.00 34.84 C ATOM 2737 CG MET C 48 −27.707 −23.431 26.014 1.00 36.90 C ATOM 2738 SD MET C 48 −29.006 −23.169 24.819 1.00 39.77 S ATOM 2739 CE MET C 48 −30.444 −23.408 25.832 1.00 37.72 C ATOM 2740 N GLY C 49 −25.787 −27.534 26.710 1.00 34.76 N ATOM 2741 CA GLY C 49 −25.736 −28.969 26.881 1.00 34.43 C ATOM 2742 C GLY C 49 −25.755 −29.614 25.502 1.00 37.75 C ATOM 2743 O GLY C 49 −25.497 −28.945 24.496 1.00 36.33 O ATOM 2744 N TRP C 50 −26.087 −30.892 25.449 1.00 35.13 N ATOM 2745 CA TRP C 50 −26.178 −31.620 24.185 1.00 34.61 C ATOM 2746 C TRP C 50 −27.186 −32.747 24.308 1.00 39.74 C ATOM 2747 O TRP C 50 −27.604 −33.081 25.417 1.00 40.68 O ATOM 2748 CB TRP C 50 −24.798 −32.163 23.758 1.00 33.36 C ATOM 2749 CG TRP C 50 −24.315 −33.329 24.572 1.00 35.59 C ATOM 2750 CD1 TRP C 50 −23.927 −33.312 25.879 1.00 38.84 C ATOM 2751 CD2 TRP C 50 −24.170 −34.685 24.128 1.00 36.15 C ATOM 2752 NE1 TRP C 50 −23.561 −34.572 26.280 1.00 39.22 N ATOM 2753 CE2 TRP C 50 −23.711 −35.439 25.229 1.00 40.58 C ATOM 2754 CE3 TRP C 50 −24.398 −35.340 22.908 1.00 37.44 C ATOM 2755 CZ2 TRP C 50 −23.456 −36.813 25.144 1.00 40.47 C ATOM 2756 CZ3 TRP C 50 −24.166 −36.707 22.829 1.00 39.61 C ATOM 2757 CH2 TRP C 50 −23.704 −37.429 23.939 1.00 41.17 C ATOM 2758 N ILE C 51 −27.582 −33.321 23.172 1.00 36.40 N ATOM 2759 CA ILE C 51 −28.504 −34.444 23.143 1.00 36.37 C ATOM 2760 C ILE C 51 −27.886 −35.575 22.345 1.00 40.89 C ATOM 2761 O ILE C 51 −27.328 −35.348 21.267 1.00 37.88 O ATOM 2762 CB ILE C 51 −29.929 −34.067 22.632 1.00 38.96 C ATOM 2763 CG1 ILE C 51 −30.880 −35.296 22.714 1.00 40.23 C ATOM 2764 CG2 ILE C 51 −29.903 −33.497 21.207 1.00 38.66 C ATOM 2765 CD1 ILE C 51 −32.294 −34.955 22.868 1.00 42.97 C ATOM 2766 N ASN C 52 −27.993 −36.803 22.887 1.00 39.50 N ATOM 2767 CA ASN C 52 −27.517 −37.998 22.221 1.00 40.16 C ATOM 2768 C ASN C 52 −28.559 −38.324 21.145 1.00 44.28 C ATOM 2769 O ASN C 52 −29.687 −38.655 21.484 1.00 45.11 O ATOM 2770 CB ASN C 52 −27.356 −39.143 23.230 1.00 40.73 C ATOM 2771 CG ASN C 52 −26.756 −40.402 22.660 1.00 48.81 C ATOM 2772 OD1 ASN C 52 −27.157 −40.886 21.593 1.00 40.43 O ATOM 2773 ND2 ASN C 52 −25.828 −41.006 23.380 1.00 44.29 N ATOM 2774 N THR C 53 −28.194 −38.208 19.866 1.00 40.01 N ATOM 2775 CA THR C 53 −29.122 −38.445 18.759 1.00 40.61 C ATOM 2776 C THR C 53 −29.458 −39.923 18.540 1.00 47.82 C ATOM 2777 O THR C 53 −30.422 −40.206 17.835 1.00 49.05 O ATOM 2778 CB THR C 53 −28.614 −37.778 17.481 1.00 42.11 C ATOM 2779 OG1 THR C 53 −27.330 −38.317 17.157 1.00 40.46 O ATOM 2780 CG2 THR C 53 −28.556 −36.254 17.619 1.00 36.13 C ATOM 2781 N ASN C 54 −28.702 −40.858 19.145 1.00 45.92 N ATOM 2782 CA ASN C 54 −28.969 −42.291 19.027 1.00 47.26 C ATOM 2783 C ASN C 54 −29.991 −42.778 20.074 1.00 52.16 C ATOM 2784 O ASN C 54 −30.904 −43.526 19.728 1.00 52.80 O ATOM 2785 CB ASN C 54 −27.677 −43.085 19.172 1.00 46.35 C ATOM 2786 CG ASN C 54 −27.825 −44.510 18.724 1.00 63.63 C ATOM 2787 OD1 ASN C 54 −28.026 −45.408 19.535 1.00 57.04 O ATOM 2788 ND2 ASN C 54 −27.739 −44.747 17.422 1.00 57.72 N ATOM 2789 N THR C 55 −29.808 −42.393 21.350 1.00 49.15 N ATOM 2790 CA THR C 55 −30.681 −42.799 22.467 1.00 49.96 C ATOM 2791 C THR C 55 −31.715 −41.737 22.858 1.00 52.55 C ATOM 2792 O THR C 55 −32.695 −42.079 23.519 1.00 53.10 O ATOM 2793 CB THR C 55 −29.839 −43.107 23.716 1.00 55.75 C ATOM 2794 OG1 THR C 55 −29.190 −41.906 24.149 1.00 52.46 O ATOM 2795 CG2 THR C 55 −28.826 −44.217 23.482 1.00 53.38 C ATOM 2796 N GLY C 56 −31.461 −40.455 22.541 1.00 47.53 N ATOM 2797 CA GLY C 56 −32.335 −39.350 22.933 1.00 46.25 C ATOM 2798 C GLY C 56 −31.983 −38.788 24.307 1.00 49.63 C ATOM 2799 O GLY C 56 −32.654 −37.852 24.733 1.00 48.46 O ATOM 2800 N GLU C 57 −30.936 −39.327 24.998 1.00 47.35 N ATOM 2801 CA GLU C 57 −30.527 −38.851 26.326 1.00 47.15 C ATOM 2802 C GLU C 57 −30.029 −37.401 26.231 1.00 48.06 C ATOM 2803 O GLU C 57 −29.088 −37.147 25.471 1.00 45.81 O ATOM 2804 CB GLU C 57 −29.398 −39.723 26.910 1.00 49.45 C ATOM 2805 CG GLU C 57 −28.947 −39.321 28.314 1.00 64.29 C ATOM 2806 CD GLU C 57 −27.633 −39.929 28.774 1.00 82.18 C ATOM 2807 OE1 GLU C 57 −27.256 −41.002 28.252 1.00 71.99 O ATOM 2808 OE2 GLU C 57 −26.986 −39.337 29.670 1.00 70.32 O ATOM 2809 N PRO C 58 −30.625 −36.469 27.023 1.00 43.61 N ATOM 2810 CA PRO C 58 −30.164 −35.082 27.019 1.00 41.79 C ATOM 2811 C PRO C 58 −29.218 −34.848 28.196 1.00 45.66 C ATOM 2812 O PRO C 58 −29.383 −35.454 29.263 1.00 43.86 O ATOM 2813 CB PRO C 58 −31.459 −34.304 27.185 1.00 42.77 C ATOM 2814 CG PRO C 58 −32.263 −35.157 28.134 1.00 48.63 C ATOM 2815 CD PRO C 58 −31.883 −36.592 27.801 1.00 45.10 C ATOM 2816 N THR C 59 −28.221 −33.981 28.001 1.00 40.90 N ATOM 2817 CA THR C 59 −27.287 −33.608 29.057 1.00 40.69 C ATOM 2818 C THR C 59 −27.391 −32.114 29.144 1.00 45.94 C ATOM 2819 O THR C 59 −27.143 −31.444 28.145 1.00 44.05 O ATOM 2820 CB THR C 59 −25.873 −34.060 28.718 1.00 48.14 C ATOM 2821 OG1 THR C 59 −25.850 −35.490 28.612 1.00 49.83 O ATOM 2822 CG2 THR C 59 −24.840 −33.583 29.745 1.00 45.08 C ATOM 2823 N TYR C 60 −27.775 −31.584 30.306 1.00 44.41 N ATOM 2824 CA TYR C 60 −27.914 −30.145 30.501 1.00 44.47 C ATOM 2825 C TYR C 60 −26.797 −29.627 31.364 1.00 51.94 C ATOM 2826 O TYR C 60 −26.498 −30.236 32.385 1.00 53.63 O ATOM 2827 CB TYR C 60 −29.240 −29.845 31.204 1.00 45.68 C ATOM 2828 CG TYR C 60 −30.431 −30.400 30.466 1.00 45.61 C ATOM 2829 CD1 TYR C 60 −30.909 −29.785 29.314 1.00 45.81 C ATOM 2830 CD2 TYR C 60 −31.038 −31.580 30.877 1.00 46.46 C ATOM 2831 CE1 TYR C 60 −31.989 −30.309 28.613 1.00 45.64 C ATOM 2832 CE2 TYR C 60 −32.123 −32.112 30.187 1.00 46.88 C ATOM 2833 CZ TYR C 60 −32.601 −31.470 29.057 1.00 47.97 C ATOM 2834 OH TYR C 60 −33.652 −31.990 28.339 1.00 44.87 O ATOM 2835 N ALA C 61 −26.227 −28.462 31.013 1.00 49.04 N ATOM 2836 CA ALA C 61 −25.216 −27.821 31.860 1.00 49.52 C ATOM 2837 C ALA C 61 −25.974 −27.254 33.079 1.00 54.51 C ATOM 2838 O ALA C 61 −27.166 −26.964 32.960 1.00 52.25 O ATOM 2839 CB ALA C 61 −24.502 −26.713 31.092 1.00 49.03 C ATOM 2840 N GLU C 62 −25.319 −27.159 34.251 1.00 55.04 N ATOM 2841 CA GLU C 62 −25.946 −26.732 35.523 1.00 56.11 C ATOM 2842 C GLU C 62 −26.909 −25.546 35.427 1.00 58.34 C ATOM 2843 O GLU C 62 −28.005 −25.610 35.988 1.00 58.41 O ATOM 2844 CB GLU C 62 −24.878 −26.431 36.598 1.00 59.36 C ATOM 2845 CG GLU C 62 −24.475 −27.642 37.422 1.00 74.93 C ATOM 2846 CD GLU C 62 −25.560 −28.214 38.315 1.00 98.93 C ATOM 2847 OE1 GLU C 62 −26.317 −27.423 38.925 1.00 87.04 O ATOM 2848 OE2 GLU C 62 −25.662 −29.460 38.394 1.00 96.62 O ATOM 2849 N GLU C 63 −26.520 −24.486 34.702 1.00 53.32 N ATOM 2850 CA GLU C 63 −27.346 −23.286 34.554 1.00 52.26 C ATOM 2851 C GLU C 63 −28.607 −23.497 33.690 1.00 54.16 C ATOM 2852 O GLU C 63 −29.485 −22.635 33.696 1.00 54.82 O ATOM 2853 CB GLU C 63 −26.509 −22.127 33.975 1.00 53.10 C ATOM 2854 CG GLU C 63 −25.405 −21.652 34.906 1.00 67.32 C ATOM 2855 CD GLU C 63 −24.459 −20.632 34.303 1.00 93.33 C ATOM 2856 OE1 GLU C 63 −24.904 −19.491 34.041 1.00 97.70 O ATOM 2857 OE2 GLU C 63 −23.263 −20.960 34.126 1.00 87.08 O ATOM 2858 N PHE C 64 −28.701 −24.618 32.951 1.00 47.53 N ATOM 2859 CA PHE C 64 −29.822 −24.918 32.066 1.00 45.54 C ATOM 2860 C PHE C 64 −30.636 −26.135 32.532 1.00 52.73 C ATOM 2861 O PHE C 64 −31.354 −26.737 31.738 1.00 51.76 O ATOM 2862 CB PHE C 64 −29.286 −25.087 30.626 1.00 44.42 C ATOM 2863 CG PHE C 64 −28.575 −23.846 30.133 1.00 43.34 C ATOM 2864 CD1 PHE C 64 −29.277 −22.826 29.511 1.00 44.21 C ATOM 2865 CD2 PHE C 64 −27.227 −23.649 30.394 1.00 44.03 C ATOM 2866 CE1 PHE C 64 −28.638 −21.645 29.125 1.00 44.23 C ATOM 2867 CE2 PHE C 64 −26.579 −22.488 29.972 1.00 45.82 C ATOM 2868 CZ PHE C 64 −27.285 −21.499 29.326 1.00 43.21 C ATOM 2869 N LYS C 65 −30.582 −26.460 33.833 1.00 53.05 N ATOM 2870 CA LYS C 65 −31.364 −27.563 34.386 1.00 54.38 C ATOM 2871 C LYS C 65 −32.677 −26.997 34.922 1.00 60.28 C ATOM 2872 O LYS C 65 −32.646 −26.027 35.682 1.00 61.57 O ATOM 2873 CB LYS C 65 −30.594 −28.270 35.517 1.00 58.48 C ATOM 2874 CG LYS C 65 −29.411 −29.071 35.006 1.00 70.31 C ATOM 2875 CD LYS C 65 −28.699 −29.850 36.095 1.00 77.70 C ATOM 2876 CE LYS C 65 −27.540 −30.631 35.525 1.00 83.81 C ATOM 2877 NZ LYS C 65 −26.829 −31.422 36.565 1.00 97.06 N ATOM 2878 N GLY C 66 −33.820 −27.585 34.527 1.00 56.71 N ATOM 2879 CA GLY C 66 −35.135 −27.178 35.027 1.00 57.44 C ATOM 2880 C GLY C 66 −36.113 −26.782 33.936 1.00 59.55 C ATOM 2881 O GLY C 66 −37.103 −27.486 33.715 1.00 60.68 O ATOM 2882 N ARG C 67 −35.852 −25.646 33.274 1.00 52.60 N ATOM 2883 CA ARG C 67 −36.747 −25.085 32.262 1.00 50.96 C ATOM 2884 C ARG C 67 −36.421 −25.479 30.817 1.00 51.95 C ATOM 2885 O ARG C 67 −37.259 −25.241 29.957 1.00 50.60 O ATOM 2886 CB ARG C 67 −36.782 −23.550 32.397 1.00 48.23 C ATOM 2887 CG ARG C 67 −37.183 −23.066 33.795 1.00 48.48 C ATOM 2888 CD ARG C 67 −37.270 −21.547 33.905 1.00 45.81 C ATOM 2889 NE ARG C 67 −35.997 −20.907 33.543 1.00 47.15 N ATOM 2890 CZ ARG C 67 −35.775 −20.114 32.491 1.00 57.65 C ATOM 2891 NH1 ARG C 67 −36.767 −19.797 31.660 1.00 40.82 N ATOM 2892 NH2 ARG C 67 −34.563 −19.614 32.276 1.00 39.93 N ATOM 2893 N PHE C 68 −35.266 −26.137 30.556 1.00 46.54 N ATOM 2894 CA PHE C 68 −34.836 −26.517 29.202 1.00 43.54 C ATOM 2895 C PHE C 68 −35.079 −28.010 28.927 1.00 46.49 C ATOM 2896 O PHE C 68 −34.752 −28.847 29.769 1.00 45.31 O ATOM 2897 CB PHE C 68 −33.351 −26.153 28.985 1.00 43.64 C ATOM 2898 CG PHE C 68 −33.079 −24.678 29.189 1.00 44.64 C ATOM 2899 CD1 PHE C 68 −32.984 −24.140 30.471 1.00 48.34 C ATOM 2900 CD2 PHE C 68 −32.999 −23.812 28.105 1.00 44.72 C ATOM 2901 CE1 PHE C 68 −32.830 −22.767 30.662 1.00 48.00 C ATOM 2902 CE2 PHE C 68 −32.821 −22.439 28.300 1.00 47.45 C ATOM 2903 CZ PHE C 68 −32.756 −21.926 29.578 1.00 46.55 C ATOM 2904 N THR C 69 −35.661 −28.338 27.746 1.00 42.97 N ATOM 2905 CA THR C 69 −35.935 −29.722 27.352 1.00 43.22 C ATOM 2906 C THR C 69 −35.421 −29.977 25.935 1.00 44.51 C ATOM 2907 O THR C 69 −35.897 −29.348 24.990 1.00 43.05 O ATOM 2908 CB THR C 69 −37.432 −30.054 27.479 1.00 50.82 C ATOM 2909 OG1 THR C 69 −37.892 −29.634 28.757 1.00 50.33 O ATOM 2910 CG2 THR C 69 −37.719 −31.541 27.326 1.00 50.08 C ATOM 2911 N PHE C 70 −34.456 −30.900 25.788 1.00 40.32 N ATOM 2912 CA PHE C 70 −33.929 −31.290 24.482 1.00 38.86 C ATOM 2913 C PHE C 70 −34.630 −32.580 24.043 1.00 45.20 C ATOM 2914 O PHE C 70 −34.671 −33.530 24.820 1.00 44.98 O ATOM 2915 CB PHE C 70 −32.407 −31.524 24.536 1.00 39.78 C ATOM 2916 CG PHE C 70 −31.544 −30.405 25.075 1.00 40.33 C ATOM 2917 CD1 PHE C 70 −31.901 −29.072 24.888 1.00 42.65 C ATOM 2918 CD2 PHE C 70 −30.340 −30.681 25.708 1.00 41.10 C ATOM 2919 CE1 PHE C 70 −31.090 −28.040 25.378 1.00 42.65 C ATOM 2920 CE2 PHE C 70 −29.536 −29.654 26.193 1.00 43.12 C ATOM 2921 CZ PHE C 70 −29.910 −28.340 26.017 1.00 41.49 C ATOM 2922 N THR C 71 −35.204 −32.607 22.823 1.00 43.02 N ATOM 2923 CA THR C 71 −35.862 −33.804 22.275 1.00 43.93 C ATOM 2924 C THR C 71 −35.455 −33.983 20.817 1.00 47.17 C ATOM 2925 O THR C 71 −34.690 −33.169 20.291 1.00 44.33 O ATOM 2926 CB THR C 71 −37.396 −33.742 22.437 1.00 50.03 C ATOM 2927 OG1 THR C 71 −37.894 −32.582 21.770 1.00 48.76 O ATOM 2928 CG2 THR C 71 −37.830 −33.748 23.891 1.00 44.13 C ATOM 2929 N LEU C 72 −35.915 −35.087 20.196 1.00 45.85 N ATOM 2930 CA LEU C 72 −35.616 −35.420 18.808 1.00 46.48 C ATOM 2931 C LEU C 72 −36.822 −35.973 18.082 1.00 52.69 C ATOM 2932 O LEU C 72 −37.747 −36.499 18.707 1.00 53.00 O ATOM 2933 CB LEU C 72 −34.582 −36.559 18.737 1.00 47.11 C ATOM 2934 CG LEU C 72 −33.302 −36.466 19.539 1.00 50.90 C ATOM 2935 CD1 LEU C 72 −32.699 −37.828 19.682 1.00 51.59 C ATOM 2936 CD2 LEU C 72 −32.297 −35.525 18.878 1.00 50.84 C ATOM 2937 N ASP C 73 −36.732 −35.958 16.743 1.00 49.46 N ATOM 2938 CA ASP C 73 −37.622 −36.664 15.826 1.00 51.42 C ATOM 2939 C ASP C 73 −36.673 −37.173 14.740 1.00 55.97 C ATOM 2940 O ASP C 73 −36.407 −36.476 13.757 1.00 55.22 O ATOM 2941 CB ASP C 73 −38.765 −35.798 15.262 1.00 53.45 C ATOM 2942 CG ASP C 73 −39.806 −36.585 14.472 1.00 63.69 C ATOM 2943 OD1 ASP C 73 −39.536 −37.759 14.127 1.00 65.24 O ATOM 2944 OD2 ASP C 73 −40.890 −36.028 14.199 1.00 68.96 O ATOM 2945 N THR C 74 −36.089 −38.358 14.986 1.00 53.14 N ATOM 2946 CA THR C 74 −35.080 −38.958 14.110 1.00 52.84 C ATOM 2947 C THR C 74 −35.614 −39.316 12.732 1.00 56.08 C ATOM 2948 O THR C 74 −34.821 −39.346 11.794 1.00 54.60 O ATOM 2949 CB THR C 74 −34.404 −40.163 14.789 1.00 57.27 C ATOM 2950 OG1 THR C 74 −35.399 −41.100 15.188 1.00 63.17 O ATOM 2951 CG2 THR C 74 −33.574 −39.757 15.993 1.00 51.44 C ATOM 2952 N SER C 75 −36.936 −39.548 12.584 1.00 54.66 N ATOM 2953 CA SER C 75 −37.542 −39.841 11.274 1.00 55.95 C ATOM 2954 C SER C 75 −37.401 −38.644 10.304 1.00 58.67 C ATOM 2955 O SER C 75 −37.318 −38.858 9.098 1.00 59.17 O ATOM 2956 CB SER C 75 −39.009 −40.248 11.419 1.00 59.94 C ATOM 2957 OG SER C 75 −39.842 −39.153 11.766 1.00 65.14 O ATOM 2958 N ILE C 76 −37.327 −37.404 10.833 1.00 53.62 N ATOM 2959 CA ILE C 76 −37.127 −36.198 10.018 1.00 52.30 C ATOM 2960 C ILE C 76 −35.811 −35.481 10.396 1.00 52.90 C ATOM 2961 O ILE C 76 −35.688 −34.286 10.139 1.00 52.08 O ATOM 2962 CB ILE C 76 −38.375 −35.265 10.064 1.00 55.25 C ATOM 2963 CG1 ILE C 76 −38.765 −34.893 11.512 1.00 54.74 C ATOM 2964 CG2 ILE C 76 −39.551 −35.936 9.346 1.00 57.07 C ATOM 2965 CD1 ILE C 76 −39.674 −33.697 11.624 1.00 59.04 C ATOM 2966 N SER C 77 −34.810 −36.228 10.941 1.00 47.96 N ATOM 2967 CA SER C 77 −33.493 −35.716 11.360 1.00 45.97 C ATOM 2968 C SER C 77 −33.567 −34.338 12.068 1.00 46.80 C ATOM 2969 O SER C 77 −32.818 −33.422 11.725 1.00 44.01 O ATOM 2970 CB SER C 77 −32.552 −35.642 10.159 1.00 48.90 C ATOM 2971 OG SER C 77 −32.426 −36.897 9.513 1.00 55.71 O ATOM 2972 N THR C 78 −34.488 −34.194 13.033 1.00 43.04 N ATOM 2973 CA THR C 78 −34.685 −32.918 13.717 1.00 41.49 C ATOM 2974 C THR C 78 −34.436 −33.027 15.224 1.00 43.81 C ATOM 2975 O THR C 78 −34.818 −34.000 15.860 1.00 43.23 O ATOM 2976 CB THR C 78 −36.075 −32.345 13.362 1.00 47.21 C ATOM 2977 OG1 THR C 78 −36.103 −32.132 11.948 1.00 44.47 O ATOM 2978 CG2 THR C 78 −36.375 −31.012 14.059 1.00 43.17 C ATOM 2979 N ALA C 79 −33.777 −32.002 15.772 1.00 40.03 N ATOM 2980 CA ALA C 79 −33.491 −31.853 17.191 1.00 39.14 C ATOM 2981 C ALA C 79 −34.285 −30.622 17.640 1.00 40.85 C ATOM 2982 O ALA C 79 −34.321 −29.621 16.915 1.00 38.22 O ATOM 2983 CB ALA C 79 −32.000 −31.621 17.401 1.00 39.04 C ATOM 2984 N TYR C 80 −34.936 −30.699 18.810 1.00 37.50 N ATOM 2985 CA TYR C 80 −35.729 −29.592 19.343 1.00 36.80 C ATOM 2986 C TYR C 80 −35.178 −29.102 20.655 1.00 41.15 C ATOM 2987 O TYR C 80 −34.569 −29.860 21.410 1.00 40.69 O ATOM 2988 CB TYR C 80 −37.183 −30.006 19.566 1.00 38.97 C ATOM 2989 CG TYR C 80 −37.867 −30.508 18.320 1.00 40.94 C ATOM 2990 CD1 TYR C 80 −38.435 −29.621 17.407 1.00 42.18 C ATOM 2991 CD2 TYR C 80 −37.946 −31.865 18.046 1.00 42.77 C ATOM 2992 CE1 TYR C 80 −39.058 −30.078 16.249 1.00 42.98 C ATOM 2993 CE2 TYR C 80 −38.568 −32.336 16.895 1.00 44.82 C ATOM 2994 CZ TYR C 80 −39.149 −31.441 16.009 1.00 52.02 C ATOM 2995 OH TYR C 80 −39.785 −31.894 14.875 1.00 55.03 O ATOM 2996 N MET C 81 −35.454 −27.842 20.952 1.00 38.31 N ATOM 2997 CA MET C 81 −35.036 −27.209 22.185 1.00 38.86 C ATOM 2998 C MET C 81 −36.239 −26.427 22.707 1.00 41.32 C ATOM 2999 O MET C 81 −36.659 −25.461 22.070 1.00 39.90 O ATOM 3000 CB MET C 81 −33.846 −26.290 21.900 1.00 40.92 C ATOM 3001 CG MET C 81 −33.025 −25.970 23.109 1.00 46.42 C ATOM 3002 SD MET C 81 −33.577 −24.533 24.001 1.00 53.17 S ATOM 3003 CE MET C 81 −33.005 −23.230 22.863 1.00 48.11 C ATOM 3004 N GLU C 82 −36.858 −26.913 23.796 1.00 37.33 N ATOM 3005 CA GLU C 82 −38.014 −26.258 24.386 1.00 37.62 C ATOM 3006 C GLU C 82 −37.588 −25.565 25.668 1.00 44.17 C ATOM 3007 O GLU C 82 −36.774 −26.093 26.417 1.00 45.34 O ATOM 3008 CB GLU C 82 −39.171 −27.238 24.636 1.00 40.33 C ATOM 3009 CG GLU C 82 −40.395 −26.570 25.248 1.00 51.38 C ATOM 3010 CD GLU C 82 −41.705 −27.317 25.105 1.00 76.33 C ATOM 3011 OE1 GLU C 82 −41.691 −28.567 25.161 1.00 74.04 O ATOM 3012 OE2 GLU C 82 −42.755 −26.648 24.973 1.00 77.13 O ATOM 3013 N LEU C 83 −38.131 −24.377 25.906 1.00 40.98 N ATOM 3014 CA LEU C 83 −37.829 −23.592 27.084 1.00 42.46 C ATOM 3015 C LEU C 83 −39.148 −23.130 27.673 1.00 48.00 C ATOM 3016 O LEU C 83 −39.932 −22.526 26.960 1.00 48.75 O ATOM 3017 CB LEU C 83 −36.944 −22.413 26.673 1.00 41.74 C ATOM 3018 CG LEU C 83 −36.536 −21.413 27.746 1.00 48.28 C ATOM 3019 CD1 LEU C 83 −36.137 −22.094 29.047 1.00 50.54 C ATOM 3020 CD2 LEU C 83 −35.436 −20.481 27.197 1.00 50.96 C ATOM 3021 N SER C 84 −39.423 −23.482 28.936 1.00 44.74 N ATOM 3022 CA SER C 84 −40.671 −23.138 29.624 1.00 46.15 C ATOM 3023 C SER C 84 −40.465 −21.954 30.571 1.00 49.18 C ATOM 3024 O SER C 84 −39.322 −21.561 30.811 1.00 47.21 O ATOM 3025 CB SER C 84 −41.176 −24.344 30.411 1.00 49.92 C ATOM 3026 OG SER C 84 −40.189 −24.773 31.333 1.00 60.20 O ATOM 3027 N SER C 85 −41.577 −21.392 31.105 1.00 46.34 N ATOM 3028 CA SER C 85 −41.566 −20.246 32.026 1.00 46.41 C ATOM 3029 C SER C 85 −40.521 −19.203 31.589 1.00 47.84 C ATOM 3030 O SER C 85 −39.610 −18.866 32.340 1.00 46.67 O ATOM 3031 CB SER C 85 −41.324 −20.717 33.459 1.00 51.60 C ATOM 3032 OG SER C 85 −42.365 −21.581 33.885 1.00 62.77 O ATOM 3033 N LEU C 86 −40.622 −18.760 30.327 1.00 44.01 N ATOM 3034 CA LEU C 86 −39.673 −17.809 29.741 1.00 42.04 C ATOM 3035 C LEU C 86 −39.483 −16.535 30.581 1.00 47.78 C ATOM 3036 O LEU C 86 −40.457 −15.984 31.097 1.00 48.21 O ATOM 3037 CB LEU C 86 −40.082 −17.445 28.302 1.00 40.04 C ATOM 3038 CG LEU C 86 −39.816 −18.512 27.241 1.00 41.12 C ATOM 3039 CD1 LEU C 86 −40.629 −18.242 25.989 1.00 38.91 C ATOM 3040 CD2 LEU C 86 −38.345 −18.569 26.870 1.00 38.83 C ATOM 3041 N ARG C 87 −38.217 −16.110 30.738 1.00 44.37 N ATOM 3042 CA ARG C 87 −37.793 −14.918 31.486 1.00 45.33 C ATOM 3043 C ARG C 87 −37.173 −13.939 30.477 1.00 47.34 C ATOM 3044 O ARG C 87 −36.679 −14.395 29.447 1.00 43.73 O ATOM 3045 CB ARG C 87 −36.691 −15.291 32.500 1.00 46.43 C ATOM 3046 CG ARG C 87 −37.122 −16.210 33.635 1.00 56.09 C ATOM 3047 CD ARG C 87 −36.368 −15.919 34.923 1.00 77.75 C ATOM 3048 NE ARG C 87 −34.995 −16.435 34.903 1.00 93.41 N ATOM 3049 CZ ARG C 87 −34.611 −17.651 35.302 1.00 109.85 C ATOM 3050 NH1 ARG C 87 −35.505 −18.530 35.754 1.00 94.20 N ATOM 3051 NH2 ARG C 87 −33.330 −18.001 35.244 1.00 95.36 N ATOM 3052 N SER C 88 −37.084 −12.628 30.806 1.00 45.15 N ATOM 3053 CA SER C 88 −36.450 −11.653 29.900 1.00 43.88 C ATOM 3054 C SER C 88 −34.957 −11.972 29.646 1.00 46.23 C ATOM 3055 O SER C 88 −34.465 −11.716 28.554 1.00 44.31 O ATOM 3056 CB SER C 88 −36.622 −10.224 30.414 1.00 48.56 C ATOM 3057 OG SER C 88 −35.889 −10.005 31.604 1.00 62.20 O ATOM 3058 N GLU C 89 −34.286 −12.630 30.618 1.00 44.67 N ATOM 3059 CA GLU C 89 −32.879 −13.049 30.535 1.00 43.02 C ATOM 3060 C GLU C 89 −32.670 −14.199 29.545 1.00 44.03 C ATOM 3061 O GLU C 89 −31.520 −14.531 29.258 1.00 42.78 O ATOM 3062 CB GLU C 89 −32.340 −13.468 31.914 1.00 45.89 C ATOM 3063 CG GLU C 89 −32.405 −12.377 32.972 1.00 57.40 C ATOM 3064 CD GLU C 89 −33.556 −12.509 33.951 1.00 82.31 C ATOM 3065 OE1 GLU C 89 −34.719 −12.338 33.520 1.00 65.11 O ATOM 3066 OE2 GLU C 89 −33.299 −12.783 35.145 1.00 88.49 O ATOM 3067 N ASP C 90 −33.759 −14.826 29.045 1.00 39.75 N ATOM 3068 CA ASP C 90 −33.675 −15.859 28.014 1.00 38.16 C ATOM 3069 C ASP C 90 −33.601 −15.215 26.629 1.00 38.48 C ATOM 3070 O ASP C 90 −33.443 −15.942 25.650 1.00 37.24 O ATOM 3071 CB ASP C 90 −34.869 −16.822 28.076 1.00 40.23 C ATOM 3072 CG ASP C 90 −35.003 −17.524 29.405 1.00 45.99 C ATOM 3073 OD1 ASP C 90 −33.969 −17.957 29.954 1.00 46.86 O ATOM 3074 OD2 ASP C 90 −36.147 −17.682 29.879 1.00 45.07 O ATOM 3075 N THR C 91 −33.726 −13.876 26.522 1.00 35.50 N ATOM 3076 CA THR C 91 −33.588 −13.185 25.245 1.00 34.04 C ATOM 3077 C THR C 91 −32.173 −13.469 24.735 1.00 36.90 C ATOM 3078 O THR C 91 −31.213 −13.192 25.446 1.00 36.84 O ATOM 3079 CB THR C 91 −33.869 −11.684 25.414 1.00 38.72 C ATOM 3080 OG1 THR C 91 −35.226 −11.536 25.849 1.00 37.88 O ATOM 3081 CG2 THR C 91 −33.604 −10.875 24.124 1.00 33.64 C ATOM 3082 N ALA C 92 −32.052 −14.080 23.550 1.00 32.89 N ATOM 3083 CA ALA C 92 −30.754 −14.492 23.003 1.00 32.23 C ATOM 3084 C ALA C 92 −30.927 −15.108 21.636 1.00 34.59 C ATOM 3085 O ALA C 92 −32.054 −15.347 21.198 1.00 33.75 O ATOM 3086 CB ALA C 92 −30.130 −15.565 23.924 1.00 33.21 C ATOM 3087 N VAL C 93 −29.796 −15.417 20.984 1.00 31.52 N ATOM 3088 CA VAL C 93 −29.760 −16.228 19.776 1.00 31.04 C ATOM 3089 C VAL C 93 −29.386 −17.640 20.285 1.00 34.81 C ATOM 3090 O VAL C 93 −28.436 −17.778 21.065 1.00 34.37 O ATOM 3091 CB VAL C 93 −28.763 −15.738 18.699 1.00 33.19 C ATOM 3092 CG1 VAL C 93 −28.569 −16.797 17.611 1.00 32.03 C ATOM 3093 CG2 VAL C 93 −29.243 −14.426 18.080 1.00 32.68 C ATOM 3094 N TYR C 94 −30.117 −18.668 19.840 1.00 31.06 N ATOM 3095 CA TYR C 94 −29.852 −20.054 20.201 1.00 30.70 C ATOM 3096 C TYR C 94 −29.388 −20.787 18.970 1.00 35.19 C ATOM 3097 O TYR C 94 −30.042 −20.687 17.936 1.00 34.82 O ATOM 3098 CB TYR C 94 −31.114 −20.716 20.768 1.00 30.69 C ATOM 3099 CG TYR C 94 −31.479 −20.138 22.111 1.00 31.16 C ATOM 3100 CD1 TYR C 94 −32.306 −19.028 22.214 1.00 32.73 C ATOM 3101 CD2 TYR C 94 −30.902 −20.629 23.276 1.00 31.99 C ATOM 3102 CE1 TYR C 94 −32.605 −18.460 23.446 1.00 33.76 C ATOM 3103 CE2 TYR C 94 −31.201 −20.077 24.519 1.00 32.65 C ATOM 3104 CZ TYR C 94 −32.051 −18.989 24.601 1.00 37.49 C ATOM 3105 OH TYR C 94 −32.313 −18.424 25.826 1.00 37.85 O ATOM 3106 N TYR C 95 −28.259 −21.513 19.066 1.00 31.75 N ATOM 3107 CA TYR C 95 −27.737 −22.291 17.948 1.00 31.12 C ATOM 3108 C TYR C 95 −27.759 −23.750 18.294 1.00 36.58 C ATOM 3109 O TYR C 95 −27.510 −24.120 19.447 1.00 35.65 O ATOM 3110 CB TYR C 95 −26.263 −21.943 17.634 1.00 32.42 C ATOM 3111 CG TYR C 95 −25.965 −20.480 17.390 1.00 33.70 C ATOM 3112 CD1 TYR C 95 −26.113 −19.917 16.128 1.00 35.21 C ATOM 3113 CD2 TYR C 95 −25.446 −19.681 18.402 1.00 34.49 C ATOM 3114 CE1 TYR C 95 −25.821 −18.576 15.894 1.00 35.70 C ATOM 3115 CE2 TYR C 95 −25.145 −18.340 18.181 1.00 34.92 C ATOM 3116 CZ TYR C 95 −25.333 −17.789 16.924 1.00 41.88 C ATOM 3117 OH TYR C 95 −25.012 −16.475 16.680 1.00 40.68 O ATOM 3118 N CYS C 96 −27.978 −24.587 17.282 1.00 34.61 N ATOM 3119 CA CYS C 96 −27.748 −26.015 17.382 1.00 35.50 C ATOM 3120 C CYS C 96 −26.408 −26.170 16.648 1.00 37.03 C ATOM 3121 O CYS C 96 −26.126 −25.405 15.716 1.00 35.03 O ATOM 3122 CB CYS C 96 −28.856 −26.836 16.729 1.00 37.04 C ATOM 3123 SG CYS C 96 −29.122 −26.492 14.973 1.00 41.53 S ATOM 3124 N ALA C 97 −25.552 −27.065 17.104 1.00 32.70 N ATOM 3125 CA ALA C 97 −24.263 −27.275 16.443 1.00 32.65 C ATOM 3126 C ALA C 97 −23.846 −28.724 16.581 1.00 37.32 C ATOM 3127 O ALA C 97 −23.861 −29.269 17.689 1.00 36.14 O ATOM 3128 CB ALA C 97 −23.205 −26.343 17.016 1.00 33.19 C ATOM 3129 N ARG C 98 −23.501 −29.364 15.455 1.00 35.53 N ATOM 3130 CA ARG C 98 −23.169 −30.787 15.460 1.00 36.66 C ATOM 3131 C ARG C 98 −21.906 −31.073 16.237 1.00 40.15 C ATOM 3132 O ARG C 98 −20.907 −30.397 16.034 1.00 40.12 O ATOM 3133 CB ARG C 98 −23.005 −31.329 14.026 1.00 35.79 C ATOM 3134 CG ARG C 98 −22.807 −32.845 13.990 1.00 38.52 C ATOM 3135 CD ARG C 98 −22.636 −33.404 12.589 1.00 38.54 C ATOM 3136 NE ARG C 98 −21.245 −33.332 12.126 1.00 38.05 N ATOM 3137 CZ ARG C 98 −20.738 −34.037 11.116 1.00 49.66 C ATOM 3138 NH1 ARG C 98 −21.499 −34.887 10.439 1.00 40.39 N ATOM 3139 NH2 ARG C 98 −19.462 −33.906 10.783 1.00 40.88 N ATOM 3140 O GLU C 99 −20.182 −34.722 16.824 1.00 39.36 O ATOM 3141 N GLU C 99 −21.923 −32.123 17.062 1.00 36.01 N ATOM 3142 CA GLU C 99 −20.726 −32.562 17.752 1.00 36.27 C ATOM 3143 C GLU C 99 −19.980 −33.514 16.790 1.00 40.48 C ATOM 3144 CB GLU C 99 −21.078 −33.238 19.099 1.00 37.68 C ATOM 3145 CG GLU C 99 −19.875 −33.508 19.991 1.00 44.26 C ATOM 3146 CD GLU C 99 −18.977 −32.335 20.350 1.00 60.17 C ATOM 3147 OE1 GLU C 99 −19.425 −31.169 20.241 1.00 45.39 O ATOM 3148 OE2 GLU C 99 −17.841 −32.591 20.811 1.00 51.81 O ATOM 3149 O GLY C 100 −16.575 −33.913 16.434 1.00 43.26 O ATOM 3150 N GLY C 100 −19.169 −32.932 15.881 1.00 37.95 N ATOM 3151 CA GLY C 100 −18.379 −33.664 14.895 1.00 38.68 C ATOM 3152 C GLY C 100 −16.913 −33.565 15.299 1.00 44.00 C ATOM 3153 O ASP C 101 −14.521 −30.532 14.281 1.00 43.47 O ATOM 3154 N ASP C 101 −16.041 −33.121 14.375 1.00 40.89 N ATOM 3155 CA ASP C 101 −14.621 −32.881 14.665 1.00 41.53 C ATOM 3156 C ASP C 101 −14.653 −31.430 15.107 1.00 44.27 C ATOM 3157 CB ASP C 101 −13.746 −33.099 13.413 1.00 44.48 C ATOM 3158 CG ASP C 101 −13.577 −34.553 13.004 1.00 52.29 C ATOM 3159 OD1 ASP C 101 −13.902 −35.445 13.821 1.00 51.22 O ATOM 3160 OD2 ASP C 101 −13.120 −34.801 11.864 1.00 58.70 O ATOM 3161 O ALA C 102 −17.404 −30.524 16.084 1.00 39.71 O ATOM 3162 N ALA C 102 −14.970 −31.221 16.409 1.00 40.87 N ATOM 3163 CA ALA C 102 −15.295 −29.940 17.052 1.00 39.67 C ATOM 3164 C ALA C 102 −16.752 −29.657 16.664 1.00 41.18 C ATOM 3165 CB ALA C 102 −14.386 −28.803 16.573 1.00 40.92 C ATOM 3166 N MET C 103 −17.258 −28.449 16.913 1.00 36.68 N ATOM 3167 CA MET C 103 −18.620 −28.119 16.494 1.00 34.23 C ATOM 3168 C MET C 103 −18.497 −27.662 15.044 1.00 38.48 C ATOM 3169 O MET C 103 −18.429 −26.474 14.750 1.00 37.28 O ATOM 3170 CB MET C 103 −19.222 −27.086 17.430 1.00 35.01 C ATOM 3171 CG MET C 103 −19.225 −27.573 18.861 1.00 37.82 C ATOM 3172 SD MET C 103 −20.069 −26.458 19.960 1.00 39.84 S ATOM 3173 CE MET C 103 −19.518 −27.119 21.566 1.00 37.25 C ATOM 3174 N ASP C 104 −18.373 −28.653 14.146 1.00 36.75 N ATOM 3175 CA ASP C 104 −18.044 −28.445 12.739 1.00 37.51 C ATOM 3176 C ASP C 104 −19.171 −27.881 11.878 1.00 40.69 C ATOM 3177 O ASP C 104 −18.870 −27.272 10.866 1.00 41.02 O ATOM 3178 CB ASP C 104 −17.461 −29.730 12.106 1.00 40.15 C ATOM 3179 CG ASP C 104 −18.243 −31.015 12.300 1.00 41.59 C ATOM 3180 OD1 ASP C 104 −19.360 −30.960 12.859 1.00 41.81 O ATOM 3181 OD2 ASP C 104 −17.732 −32.080 11.909 1.00 45.34 O ATOM 3182 N TYR C 105 −20.433 −28.066 12.252 1.00 38.20 N ATOM 3183 CA TYR C 105 −21.558 −27.501 11.506 1.00 37.29 C ATOM 3184 C TYR C 105 −22.482 −26.830 12.500 1.00 39.73 C ATOM 3185 O TYR C 105 −22.862 −27.450 13.483 1.00 38.23 O ATOM 3186 CB TYR C 105 −22.299 −28.582 10.708 1.00 39.27 C ATOM 3187 CG TYR C 105 −21.439 −29.141 9.606 1.00 43.09 C ATOM 3188 CD1 TYR C 105 −21.252 −28.441 8.419 1.00 45.85 C ATOM 3189 CD2 TYR C 105 −20.722 −30.319 9.789 1.00 44.77 C ATOM 3190 CE1 TYR C 105 −20.408 −28.922 7.420 1.00 48.74 C ATOM 3191 CE2 TYR C 105 −19.864 −30.802 8.805 1.00 47.26 C ATOM 3192 CZ TYR C 105 −19.708 −30.101 7.618 1.00 55.46 C ATOM 3193 OH TYR C 105 −18.870 −30.560 6.624 1.00 57.88 O ATOM 3194 N TRP C 106 −22.796 −25.554 12.270 1.00 35.79 N ATOM 3195 CA TRP C 106 −23.669 −24.770 13.131 1.00 34.32 C ATOM 3196 C TRP C 106 −24.925 −24.425 12.381 1.00 38.63 C ATOM 3197 O TRP C 106 −24.862 −24.157 11.177 1.00 39.38 O ATOM 3198 CB TRP C 106 −22.974 −23.459 13.527 1.00 32.65 C ATOM 3199 CG TRP C 106 −21.848 −23.643 14.486 1.00 33.67 C ATOM 3200 CD1 TRP C 106 −20.643 −24.225 14.230 1.00 37.22 C ATOM 3201 CD2 TRP C 106 −21.779 −23.142 15.825 1.00 33.03 C ATOM 3202 NE1 TRP C 106 −19.856 −24.190 15.354 1.00 36.90 N ATOM 3203 CE2 TRP C 106 −20.526 −23.526 16.350 1.00 37.50 C ATOM 3204 CE3 TRP C 106 −22.672 −22.442 16.649 1.00 33.13 C ATOM 3205 CZ2 TRP C 106 −20.139 −23.224 17.659 1.00 36.59 C ATOM 3206 CZ3 TRP C 106 −22.293 −22.153 17.952 1.00 34.77 C ATOM 3207 CH2 TRP C 106 −21.039 −22.540 18.444 1.00 36.16 C ATOM 3208 N GLY C 107 −26.057 −24.338 13.093 1.00 35.05 N ATOM 3209 CA GLY C 107 −27.302 −23.858 12.496 1.00 34.89 C ATOM 3210 C GLY C 107 −27.139 −22.326 12.324 1.00 38.88 C ATOM 3211 O GLY C 107 −26.186 −21.751 12.863 1.00 37.72 O ATOM 3212 N GLN C 108 −28.033 −21.674 11.562 1.00 35.72 N ATOM 3213 CA GLN C 108 −27.940 −20.218 11.349 1.00 34.48 C ATOM 3214 C GLN C 108 −28.236 −19.417 12.623 1.00 36.52 C ATOM 3215 O GLN C 108 −27.866 −18.249 12.708 1.00 35.28 O ATOM 3216 CB GLN C 108 −28.813 −19.748 10.165 1.00 36.25 C ATOM 3217 CG GLN C 108 −30.320 −19.534 10.427 1.00 37.04 C ATOM 3218 CD GLN C 108 −31.133 −20.801 10.402 1.00 42.51 C ATOM 3219 OE1 GLN C 108 −30.613 −21.915 10.552 1.00 40.06 O ATOM 3220 NE2 GLN C 108 −32.439 −20.672 10.236 1.00 31.88 N ATOM 3221 N GLY C 109 −28.898 −20.044 13.606 1.00 33.66 N ATOM 3222 CA GLY C 109 −29.227 −19.415 14.874 1.00 33.01 C ATOM 3223 C GLY C 109 −30.686 −19.012 14.839 1.00 36.49 C ATOM 3224 O GLY C 109 −31.227 −18.754 13.761 1.00 36.95 O ATOM 3225 N THR C 110 −31.333 −19.007 16.003 1.00 32.70 N ATOM 3226 CA THR C 110 −32.728 −18.594 16.144 1.00 32.46 C ATOM 3227 C THR C 110 −32.788 −17.523 17.217 1.00 34.68 C ATOM 3228 O THR C 110 −32.420 −17.797 18.356 1.00 33.77 O ATOM 3229 CB THR C 110 −33.633 −19.782 16.496 1.00 40.27 C ATOM 3230 OG1 THR C 110 −33.657 −20.692 15.393 1.00 36.94 O ATOM 3231 CG2 THR C 110 −35.063 −19.350 16.824 1.00 36.82 C ATOM 3232 N THR C 111 −33.266 −16.320 16.863 1.00 30.46 N ATOM 3233 CA THR C 111 −33.426 −15.225 17.830 1.00 30.94 C ATOM 3234 C THR C 111 −34.693 −15.485 18.616 1.00 34.43 C ATOM 3235 O THR C 111 −35.740 −15.734 18.022 1.00 35.46 O ATOM 3236 CB THR C 111 −33.559 −13.850 17.128 1.00 35.59 C ATOM 3237 OG1 THR C 111 −32.469 −13.673 16.229 1.00 36.40 O ATOM 3238 CG2 THR C 111 −33.572 −12.686 18.115 1.00 34.29 C ATOM 3239 N VAL C 112 −34.607 −15.402 19.930 1.00 30.68 N ATOM 3240 CA VAL C 112 −35.761 −15.543 20.811 1.00 31.81 C ATOM 3241 C VAL C 112 −35.812 −14.269 21.639 1.00 37.05 C ATOM 3242 O VAL C 112 −34.865 −13.998 22.371 1.00 37.54 O ATOM 3243 CB VAL C 112 −35.666 −16.790 21.725 1.00 35.23 C ATOM 3244 CG1 VAL C 112 −36.822 −16.834 22.728 1.00 35.87 C ATOM 3245 CG2 VAL C 112 −35.610 −18.081 20.906 1.00 34.03 C ATOM 3246 N THR C 113 −36.904 −13.497 21.536 1.00 34.53 N ATOM 3247 CA THR C 113 −37.098 −12.286 22.337 1.00 34.18 C ATOM 3248 C THR C 113 −38.182 −12.558 23.369 1.00 37.78 C ATOM 3249 O THR C 113 −39.293 −12.942 22.998 1.00 36.82 O ATOM 3250 CB THR C 113 −37.485 −11.096 21.457 1.00 37.79 C ATOM 3251 OG1 THR C 113 −36.453 −10.895 20.497 1.00 34.63 O ATOM 3252 CG2 THR C 113 −37.681 −9.804 22.269 1.00 36.47 C ATOM 3253 N VAL C 114 −37.867 −12.345 24.657 1.00 35.36 N ATOM 3254 CA VAL C 114 −38.836 −12.507 25.743 1.00 36.70 C ATOM 3255 C VAL C 114 −39.070 −11.115 26.326 1.00 41.49 C ATOM 3256 O VAL C 114 −38.121 −10.477 26.790 1.00 41.38 O ATOM 3257 CB VAL C 114 −38.376 −13.514 26.821 1.00 40.57 C ATOM 3258 CG1 VAL C 114 −39.460 −13.700 27.882 1.00 41.97 C ATOM 3259 CG2 VAL C 114 −37.999 −14.855 26.189 1.00 39.76 C ATOM 3260 N SER C 115 −40.323 −10.642 26.289 1.00 37.44 N ATOM 3261 CA SER C 115 −40.650 −9.318 26.789 1.00 37.71 C ATOM 3262 C SER C 115 −42.138 −9.137 27.039 1.00 44.81 C ATOM 3263 O SER C 115 −42.973 −9.742 26.374 1.00 45.12 O ATOM 3264 CB SER C 115 −40.180 −8.255 25.799 1.00 36.58 C ATOM 3265 OG SER C 115 −40.711 −6.980 26.119 1.00 45.95 O ATOM 3266 N SER C 116 −42.453 −8.250 27.971 1.00 43.52 N ATOM 3267 CA SER C 116 −43.817 −7.862 28.299 1.00 45.24 C ATOM 3268 C SER C 116 −44.396 −6.913 27.224 1.00 47.63 C ATOM 3269 O SER C 116 −45.613 −6.738 27.173 1.00 48.53 O ATOM 3270 CB SER C 116 −43.833 −7.150 29.644 1.00 50.86 C ATOM 3271 OG SER C 116 −45.164 −6.936 30.077 1.00 71.06 O ATOM 3272 N ALA C 117 −43.532 −6.272 26.399 1.00 42.07 N ATOM 3273 CA ALA C 117 −43.971 −5.331 25.359 1.00 41.82 C ATOM 3274 C ALA C 117 −44.864 −5.969 24.297 1.00 46.42 C ATOM 3275 O ALA C 117 −44.794 −7.171 24.044 1.00 45.12 O ATOM 3276 CB ALA C 117 −42.773 −4.679 24.685 1.00 40.90 C ATOM 3277 N SER C 118 −45.711 −5.140 23.693 1.00 45.26 N ATOM 3278 CA SER C 118 −46.643 −5.541 22.648 1.00 45.54 C ATOM 3279 C SER C 118 −46.135 −5.030 21.311 1.00 45.34 C ATOM 3280 O SER C 118 −45.396 −4.048 21.266 1.00 43.82 O ATOM 3281 CB SER C 118 −48.030 −4.970 22.931 1.00 51.75 C ATOM 3282 OG SER C 118 −48.564 −5.534 24.118 1.00 64.79 O ATOM 3283 N THR C 119 −46.530 −5.706 20.224 1.00 40.43 N ATOM 3284 CA THR C 119 −46.141 −5.338 18.862 1.00 37.93 C ATOM 3285 C THR C 119 −46.515 −3.880 18.584 1.00 41.88 C ATOM 3286 O THR C 119 −47.667 −3.494 18.805 1.00 43.45 O ATOM 3287 CB THR C 119 −46.814 −6.283 17.848 1.00 41.22 C ATOM 3288 OG1 THR C 119 −46.490 −7.624 18.206 1.00 40.13 O ATOM 3289 CG2 THR C 119 −46.390 −6.010 16.401 1.00 34.48 C ATOM 3290 N LYS C 120 −45.542 −3.074 18.132 1.00 36.14 N ATOM 3291 CA LYS C 120 −45.779 −1.670 17.809 1.00 36.50 C ATOM 3292 C LYS C 120 −44.846 −1.208 16.692 1.00 39.05 C ATOM 3293 O LYS C 120 −43.648 −1.455 16.766 1.00 39.04 O ATOM 3294 CB LYS C 120 −45.584 −0.780 19.048 1.00 37.54 C ATOM 3295 CG LYS C 120 −46.175 0.615 18.845 1.00 39.66 C ATOM 3296 CD LYS C 120 −45.645 1.638 19.823 1.00 35.79 C ATOM 3297 CE LYS C 120 −45.912 3.051 19.361 1.00 36.99 C ATOM 3298 NZ LYS C 120 −44.934 3.522 18.337 1.00 42.09 N ATOM 3299 N GLY C 121 −45.387 −0.522 15.681 1.00 35.36 N ATOM 3300 CA GLY C 121 −44.591 0.019 14.583 1.00 33.69 C ATOM 3301 C GLY C 121 −43.882 1.303 15.055 1.00 36.60 C ATOM 3302 O GLY C 121 −44.329 1.922 16.014 1.00 35.09 O ATOM 3303 N PRO C 122 −42.780 1.695 14.403 1.00 33.96 N ATOM 3304 CA PRO C 122 −42.072 2.915 14.815 1.00 33.88 C ATOM 3305 C PRO C 122 −42.771 4.217 14.414 1.00 39.19 C ATOM 3306 O PRO C 122 −43.578 4.246 13.489 1.00 38.22 O ATOM 3307 CB PRO C 122 −40.739 2.828 14.054 1.00 34.32 C ATOM 3308 CG PRO C 122 −41.064 2.022 12.806 1.00 37.97 C ATOM 3309 CD PRO C 122 −42.127 1.038 13.243 1.00 34.54 C ATOM 3310 N SER C 123 −42.409 5.299 15.104 1.00 37.89 N ATOM 3311 CA SER C 123 −42.766 6.659 14.727 1.00 38.91 C ATOM 3312 C SER C 123 −41.485 7.111 14.055 1.00 41.92 C ATOM 3313 O SER C 123 −40.405 6.842 14.589 1.00 42.38 O ATOM 3314 CB SER C 123 −43.061 7.533 15.943 1.00 44.48 C ATOM 3315 OG SER C 123 −44.341 7.237 16.473 1.00 55.10 O ATOM 3316 N VAL C 124 −41.572 7.719 12.866 1.00 37.46 N ATOM 3317 CA VAL C 124 −40.384 8.138 12.128 1.00 35.79 C ATOM 3318 C VAL C 124 −40.343 9.655 12.076 1.00 41.49 C ATOM 3319 O VAL C 124 −41.287 10.282 11.602 1.00 42.53 O ATOM 3320 CB VAL C 124 −40.322 7.489 10.723 1.00 37.10 C ATOM 3321 CG1 VAL C 124 −39.030 7.869 10.008 1.00 35.76 C ATOM 3322 CG2 VAL C 124 −40.433 5.971 10.841 1.00 35.79 C ATOM 3323 N PHE C 125 −39.252 10.237 12.587 1.00 37.30 N ATOM 3324 CA PHE C 125 −39.049 11.677 12.649 1.00 36.95 C ATOM 3325 C PHE C 125 −37.808 12.042 11.840 1.00 40.08 C ATOM 3326 O PHE C 125 −36.850 11.278 11.837 1.00 37.20 O ATOM 3327 CB PHE C 125 −38.854 12.103 14.116 1.00 38.71 C ATOM 3328 CG PHE C 125 −39.985 11.673 15.020 1.00 39.28 C ATOM 3329 CD1 PHE C 125 −41.274 12.145 14.818 1.00 41.37 C ATOM 3330 CD2 PHE C 125 −39.759 10.804 16.079 1.00 40.99 C ATOM 3331 CE1 PHE C 125 −42.325 11.728 15.638 1.00 42.44 C ATOM 3332 CE2 PHE C 125 −40.804 10.410 16.919 1.00 43.79 C ATOM 3333 CZ PHE C 125 −42.075 10.895 16.706 1.00 42.60 C ATOM 3334 N PRO C 126 −37.795 13.212 11.181 1.00 39.92 N ATOM 3335 CA PRO C 126 −36.608 13.602 10.420 1.00 39.47 C ATOM 3336 C PRO C 126 −35.514 14.166 11.326 1.00 40.00 C ATOM 3337 O PRO C 126 −35.806 14.769 12.360 1.00 39.10 O ATOM 3338 CB PRO C 126 −37.143 14.700 9.484 1.00 42.36 C ATOM 3339 CG PRO C 126 −38.200 15.362 10.284 1.00 47.50 C ATOM 3340 CD PRO C 126 −38.833 14.264 11.125 1.00 42.41 C ATOM 3341 N LEU C 127 −34.255 13.975 10.909 1.00 35.97 N ATOM 3342 CA LEU C 127 −33.065 14.574 11.516 1.00 35.80 C ATOM 3343 C LEU C 127 −32.630 15.558 10.413 1.00 41.86 C ATOM 3344 O LEU C 127 −31.912 15.193 9.476 1.00 39.52 O ATOM 3345 CB LEU C 127 −31.980 13.532 11.819 1.00 34.86 C ATOM 3346 CG LEU C 127 −32.335 12.473 12.869 1.00 36.48 C ATOM 3347 CD1 LEU C 127 −31.303 11.375 12.882 1.00 34.90 C ATOM 3348 CD2 LEU C 127 −32.428 13.093 14.265 1.00 36.49 C ATOM 3349 N ALA C 128 −33.200 16.769 10.454 1.00 40.92 N ATOM 3350 CA ALA C 128 −33.007 17.769 9.396 1.00 42.03 C ATOM 3351 C ALA C 128 −31.552 18.225 9.221 1.00 46.13 C ATOM 3352 O ALA C 128 −30.868 18.419 10.221 1.00 45.47 O ATOM 3353 CB ALA C 128 −33.900 18.974 9.656 1.00 44.04 C ATOM 3354 N PRO C 129 −31.082 18.428 7.968 1.00 45.18 N ATOM 3355 CA PRO C 129 −29.709 18.909 7.770 1.00 48.35 C ATOM 3356 C PRO C 129 −29.532 20.332 8.302 1.00 59.59 C ATOM 3357 O PRO C 129 −30.449 21.147 8.180 1.00 59.01 O ATOM 3358 CB PRO C 129 −29.510 18.845 6.249 1.00 49.63 C ATOM 3359 CG PRO C 129 −30.860 18.885 5.688 1.00 52.25 C ATOM 3360 CD PRO C 129 −31.770 18.234 6.677 1.00 46.36 C ATOM 3361 N SER C 130 −28.365 20.618 8.906 1.00 63.13 N ATOM 3362 CA SER C 130 −28.063 21.940 9.470 1.00 67.28 C ATOM 3363 C SER C 130 −27.376 22.840 8.440 1.00 76.61 C ATOM 3364 O SER C 130 −27.081 22.400 7.323 1.00 76.81 O ATOM 3365 CB SER C 130 −27.193 21.805 10.722 1.00 73.73 C ATOM 3366 OG SER C 130 −25.802 21.860 10.440 1.00 87.12 O ATOM 3367 N SER C 131 −27.117 24.106 8.834 1.00 76.52 N ATOM 3368 CA SER C 131 −26.457 25.118 7.999 1.00 78.64 C ATOM 3369 C SER C 131 −25.328 25.793 8.782 1.00 84.72 C ATOM 3370 O SER C 131 −24.161 25.684 8.404 1.00 85.42 O ATOM 3371 CB SER C 131 −27.469 26.164 7.540 1.00 82.74 C ATOM 3372 OG SER C 131 −28.231 26.661 8.629 1.00 92.26 O ATOM 3373 N GLY C 137 −19.451 22.330 3.155 1.00 70.36 N ATOM 3374 CA GLY C 137 −19.227 21.411 2.042 1.00 69.22 C ATOM 3375 C GLY C 137 −20.110 20.163 2.136 1.00 68.93 C ATOM 3376 O GLY C 137 −20.907 19.916 1.232 1.00 67.73 O ATOM 3377 N THR C 138 −19.989 19.395 3.235 1.00 62.57 N ATOM 3378 CA THR C 138 −20.756 18.156 3.435 1.00 59.19 C ATOM 3379 C THR C 138 −21.771 18.336 4.580 1.00 59.81 C ATOM 3380 O THR C 138 −21.435 18.897 5.623 1.00 60.22 O ATOM 3381 CB THR C 138 −19.820 16.922 3.591 1.00 67.18 C ATOM 3382 OG1 THR C 138 −20.572 15.804 4.071 1.00 70.05 O ATOM 3383 CG2 THR C 138 −18.647 17.165 4.519 1.00 66.68 C ATOM 3384 N ALA C 139 −23.018 17.870 4.366 1.00 53.15 N ATOM 3385 CA ALA C 139 −24.111 17.970 5.337 1.00 50.79 C ATOM 3386 C ALA C 139 −24.601 16.590 5.758 1.00 49.65 C ATOM 3387 O ALA C 139 −24.678 15.695 4.925 1.00 48.16 O ATOM 3388 CB ALA C 139 −25.268 18.746 4.731 1.00 51.34 C ATOM 3389 N ALA C 140 −24.965 16.430 7.036 1.00 43.44 N ATOM 3390 CA ALA C 140 −25.513 15.172 7.548 1.00 41.09 C ATOM 3391 C ALA C 140 −27.009 15.332 7.759 1.00 42.87 C ATOM 3392 O ALA C 140 −27.457 16.384 8.185 1.00 42.46 O ATOM 3393 CB ALA C 140 −24.853 14.796 8.864 1.00 41.79 C ATOM 3394 N LEU C 141 −27.779 14.302 7.445 1.00 39.12 N ATOM 3395 CA LEU C 141 −29.219 14.285 7.670 1.00 38.24 C ATOM 3396 C LEU C 141 −29.595 12.854 8.031 1.00 42.42 C ATOM 3397 O LEU C 141 −28.747 11.963 7.947 1.00 43.71 O ATOM 3398 CB LEU C 141 −29.999 14.805 6.443 1.00 38.37 C ATOM 3399 CG LEU C 141 −29.873 13.998 5.131 1.00 42.64 C ATOM 3400 CD1 LEU C 141 −31.098 13.129 4.892 1.00 41.82 C ATOM 3401 CD2 LEU C 141 −29.684 14.920 3.933 1.00 45.29 C ATOM 3402 N GLY C 142 −30.826 12.623 8.454 1.00 36.49 N ATOM 3403 CA GLY C 142 −31.201 11.272 8.819 1.00 35.92 C ATOM 3404 C GLY C 142 −32.652 11.143 9.235 1.00 39.79 C ATOM 3405 O GLY C 142 −33.435 12.077 9.071 1.00 39.99 O ATOM 3406 N CYS C 143 −32.994 9.984 9.771 1.00 36.21 N ATOM 3407 CA CYS C 143 −34.328 9.680 10.278 1.00 37.37 C ATOM 3408 C CYS C 143 −34.159 9.019 11.632 1.00 39.29 C ATOM 3409 O CYS C 143 −33.326 8.126 11.753 1.00 38.92 O ATOM 3410 CB CYS C 143 −35.079 8.759 9.321 1.00 38.59 C ATOM 3411 SG CYS C 143 −35.870 9.604 7.927 1.00 44.04 S ATOM 3412 N LEU C 144 −34.954 9.430 12.629 1.00 34.45 N ATOM 3413 CA LEU C 144 −34.971 8.825 13.957 1.00 33.07 C ATOM 3414 C LEU C 144 −36.175 7.882 13.945 1.00 35.80 C ATOM 3415 O LEU C 144 −37.301 8.337 13.732 1.00 34.17 O ATOM 3416 CB LEU C 144 −35.105 9.907 15.056 1.00 33.74 C ATOM 3417 CG LEU C 144 −35.398 9.423 16.496 1.00 36.68 C ATOM 3418 CD1 LEU C 144 −34.327 8.469 16.995 1.00 36.40 C ATOM 3419 CD2 LEU C 144 −35.520 10.608 17.456 1.00 37.03 C ATOM 3420 N VAL C 145 −35.922 6.567 14.098 1.00 32.86 N ATOM 3421 CA VAL C 145 −36.933 5.503 14.089 1.00 31.94 C ATOM 3422 C VAL C 145 −37.173 5.126 15.550 1.00 36.81 C ATOM 3423 O VAL C 145 −36.453 4.301 16.116 1.00 34.76 O ATOM 3424 CB VAL C 145 −36.439 4.301 13.245 1.00 34.65 C ATOM 3425 CG1 VAL C 145 −37.533 3.249 13.121 1.00 33.48 C ATOM 3426 CG2 VAL C 145 −35.974 4.769 11.858 1.00 34.52 C ATOM 3427 N LYS C 146 −38.204 5.715 16.147 1.00 35.25 N ATOM 3428 CA LYS C 146 −38.459 5.611 17.573 1.00 35.58 C ATOM 3429 C LYS C 146 −39.548 4.640 18.025 1.00 38.97 C ATOM 3430 O LYS C 146 −40.602 4.553 17.402 1.00 40.05 O ATOM 3431 CB LYS C 146 −38.803 7.023 18.114 1.00 39.35 C ATOM 3432 CG LYS C 146 −37.801 7.567 19.131 1.00 48.41 C ATOM 3433 CD LYS C 146 −38.452 8.299 20.251 1.00 54.56 C ATOM 3434 CE LYS C 146 −37.530 8.445 21.435 1.00 54.44 C ATOM 3435 NZ LYS C 146 −37.415 7.195 22.225 1.00 59.15 N ATOM 3436 N ASP C 147 −39.309 3.991 19.187 1.00 32.81 N ATOM 3437 CA ASP C 147 −40.278 3.178 19.926 1.00 32.65 C ATOM 3438 C ASP C 147 −41.031 2.108 19.118 1.00 36.32 C ATOM 3439 O ASP C 147 −42.230 2.223 18.885 1.00 38.15 O ATOM 3440 CB ASP C 147 −41.283 4.133 20.601 1.00 34.77 C ATOM 3441 CG ASP C 147 −40.648 5.156 21.525 1.00 43.50 C ATOM 3442 OD1 ASP C 147 −39.502 4.933 21.963 1.00 43.44 O ATOM 3443 OD2 ASP C 147 −41.301 6.177 21.812 1.00 47.34 O ATOM 3444 N TYR C 148 −40.340 1.055 18.733 1.00 31.65 N ATOM 3445 CA TYR C 148 −40.958 −0.058 18.004 1.00 30.87 C ATOM 3446 C TYR C 148 −40.642 −1.348 18.724 1.00 32.58 C ATOM 3447 O TYR C 148 −39.721 −1.401 19.531 1.00 31.62 O ATOM 3448 CB TYR C 148 −40.492 −0.119 16.539 1.00 29.90 C ATOM 3449 CG TYR C 148 −39.010 −0.366 16.368 1.00 30.49 C ATOM 3450 CD1 TYR C 148 −38.510 −1.656 16.218 1.00 30.97 C ATOM 3451 CD2 TYR C 148 −38.109 0.696 16.310 1.00 31.12 C ATOM 3452 CE1 TYR C 148 −37.146 −1.888 16.060 1.00 30.32 C ATOM 3453 CE2 TYR C 148 −36.747 0.478 16.120 1.00 30.49 C ATOM 3454 CZ TYR C 148 −36.264 −0.818 16.031 1.00 36.71 C ATOM 3455 OH TYR C 148 −34.926 −1.044 15.829 1.00 34.36 O ATOM 3456 N PHE C 149 −41.430 −2.374 18.451 1.00 29.78 N ATOM 3457 CA PHE C 149 −41.247 −3.685 19.045 1.00 28.55 C ATOM 3458 C PHE C 149 −41.977 −4.700 18.158 1.00 34.99 C ATOM 3459 O PHE C 149 −43.120 −4.441 17.777 1.00 35.21 O ATOM 3460 CB PHE C 149 −41.803 −3.733 20.500 1.00 30.35 C ATOM 3461 CG PHE C 149 −41.651 −5.089 21.147 1.00 31.30 C ATOM 3462 CD1 PHE C 149 −40.484 −5.429 21.822 1.00 32.84 C ATOM 3463 CD2 PHE C 149 −42.626 −6.068 20.987 1.00 33.45 C ATOM 3464 CE1 PHE C 149 −40.297 −6.720 22.321 1.00 33.55 C ATOM 3465 CE2 PHE C 149 −42.440 −7.357 21.492 1.00 35.85 C ATOM 3466 CZ PHE C 149 −41.283 −7.672 22.165 1.00 33.11 C ATOM 3467 N PRO C 150 −41.363 −5.862 17.868 1.00 32.34 N ATOM 3468 CA PRO C 150 −40.010 −6.326 18.211 1.00 31.46 C ATOM 3469 C PRO C 150 −39.048 −5.869 17.117 1.00 35.48 C ATOM 3470 O PRO C 150 −39.430 −5.138 16.194 1.00 33.77 O ATOM 3471 CB PRO C 150 −40.172 −7.858 18.192 1.00 33.13 C ATOM 3472 CG PRO C 150 −41.212 −8.076 17.132 1.00 37.85 C ATOM 3473 CD PRO C 150 −42.195 −6.972 17.374 1.00 34.21 C ATOM 3474 N GLU C 151 −37.819 −6.366 17.172 1.00 31.95 N ATOM 3475 CA GLU C 151 −36.885 −6.169 16.081 1.00 30.85 C ATOM 3476 C GLU C 151 −37.346 −7.111 14.938 1.00 33.62 C ATOM 3477 O GLU C 151 −38.072 −8.065 15.207 1.00 32.36 O ATOM 3478 CB GLU C 151 −35.468 −6.521 16.529 1.00 31.57 C ATOM 3479 CG GLU C 151 −34.927 −5.542 17.553 1.00 36.89 C ATOM 3480 CD GLU C 151 −33.419 −5.433 17.528 1.00 55.05 C ATOM 3481 OE1 GLU C 151 −32.899 −4.717 16.642 1.00 43.17 O ATOM 3482 OE2 GLU C 151 −32.757 −6.131 18.330 1.00 50.96 O ATOM 3483 N PRO C 152 −36.952 −6.860 13.681 1.00 30.67 N ATOM 3484 CA PRO C 152 −36.103 −5.778 13.207 1.00 29.57 C ATOM 3485 C PRO C 152 −36.886 −4.715 12.473 1.00 34.73 C ATOM 3486 O PRO C 152 −38.026 −4.931 12.058 1.00 34.13 O ATOM 3487 CB PRO C 152 −35.162 −6.500 12.240 1.00 31.06 C ATOM 3488 CG PRO C 152 −36.071 −7.528 11.577 1.00 35.02 C ATOM 3489 CD PRO C 152 −37.055 −7.943 12.667 1.00 31.45 C ATOM 3490 N VAL C 153 −36.230 −3.581 12.284 1.00 33.27 N ATOM 3491 CA VAL C 153 −36.676 −2.464 11.466 1.00 34.10 C ATOM 3492 C VAL C 153 −35.628 −2.419 10.355 1.00 36.23 C ATOM 3493 O VAL C 153 −34.461 −2.727 10.615 1.00 36.00 O ATOM 3494 CB VAL C 153 −36.749 −1.137 12.293 1.00 39.53 C ATOM 3495 CG1 VAL C 153 −35.885 −0.021 11.708 1.00 40.72 C ATOM 3496 CG2 VAL C 153 −38.186 −0.652 12.433 1.00 39.76 C ATOM 3497 N THR C 154 −36.017 −2.040 9.142 1.00 32.69 N ATOM 3498 CA THR C 154 −35.055 −1.852 8.055 1.00 32.45 C ATOM 3499 C THR C 154 −35.212 −0.447 7.522 1.00 34.76 C ATOM 3500 O THR C 154 −36.319 0.091 7.526 1.00 34.11 O ATOM 3501 CB THR C 154 −35.229 −2.890 6.938 1.00 37.98 C ATOM 3502 OG1 THR C 154 −36.540 −2.768 6.405 1.00 35.51 O ATOM 3503 CG2 THR C 154 −34.983 −4.304 7.412 1.00 36.93 C ATOM 3504 N VAL C 155 −34.112 0.147 7.055 1.00 31.22 N ATOM 3505 CA VAL C 155 −34.135 1.494 6.488 1.00 31.26 C ATOM 3506 C VAL C 155 −33.399 1.498 5.170 1.00 35.98 C ATOM 3507 O VAL C 155 −32.309 0.939 5.073 1.00 38.41 O ATOM 3508 CB VAL C 155 −33.529 2.578 7.419 1.00 34.20 C ATOM 3509 CG1 VAL C 155 −33.774 3.986 6.850 1.00 34.32 C ATOM 3510 CG2 VAL C 155 −34.081 2.462 8.831 1.00 33.57 C ATOM 3511 N SER C 156 −33.981 2.136 4.168 1.00 31.85 N ATOM 3512 CA SER C 156 −33.329 2.390 2.889 1.00 31.67 C ATOM 3513 C SER C 156 −33.532 3.890 2.648 1.00 35.07 C ATOM 3514 O SER C 156 −34.326 4.511 3.358 1.00 32.90 O ATOM 3515 CB SER C 156 −33.909 1.527 1.763 1.00 33.56 C ATOM 3516 OG SER C 156 −35.139 1.994 1.233 1.00 38.04 O ATOM 3517 N TRP C 157 −32.798 4.469 1.696 1.00 33.68 N ATOM 3518 CA TRP C 157 −32.892 5.887 1.354 1.00 34.59 C ATOM 3519 C TRP C 157 −33.226 6.021 −0.124 1.00 40.18 C ATOM 3520 O TRP C 157 −32.605 5.344 −0.956 1.00 40.14 O ATOM 3521 CB TRP C 157 −31.588 6.599 1.694 1.00 33.76 C ATOM 3522 CG TRP C 157 −31.442 6.848 3.163 1.00 34.18 C ATOM 3523 CD1 TRP C 157 −30.921 6.004 4.099 1.00 36.15 C ATOM 3524 CD2 TRP C 157 −31.921 7.992 3.872 1.00 33.96 C ATOM 3525 NE1 TRP C 157 −31.019 6.568 5.350 1.00 35.46 N ATOM 3526 CE2 TRP C 157 −31.641 7.786 5.239 1.00 37.15 C ATOM 3527 CE3 TRP C 157 −32.553 9.186 3.478 1.00 35.25 C ATOM 3528 CZ2 TRP C 157 −31.972 8.725 6.215 1.00 35.97 C ATOM 3529 CZ3 TRP C 157 −32.889 10.111 4.452 1.00 36.69 C ATOM 3530 CH2 TRP C 157 −32.567 9.891 5.796 1.00 36.63 C ATOM 3531 N ASN C 158 −34.244 6.845 −0.448 1.00 37.21 N ATOM 3532 CA ASN C 158 −34.702 7.060 −1.831 1.00 38.69 C ATOM 3533 C ASN C 158 −35.024 5.740 −2.555 1.00 42.14 C ATOM 3534 O ASN C 158 −34.682 5.562 −3.729 1.00 42.39 O ATOM 3535 CB ASN C 158 −33.682 7.917 −2.604 1.00 40.22 C ATOM 3536 CG ASN C 158 −33.522 9.295 −2.008 1.00 46.21 C ATOM 3537 OD1 ASN C 158 −34.342 9.733 −1.212 1.00 40.14 O ATOM 3538 ND2 ASN C 158 −32.487 10.024 −2.400 1.00 38.65 N ATOM 3539 N SER C 159 −35.668 4.807 −1.818 1.00 37.79 N ATOM 3540 CA SER C 159 −36.079 3.491 −2.301 1.00 38.40 C ATOM 3541 C SER C 159 −34.904 2.654 −2.844 1.00 41.77 C ATOM 3542 O SER C 159 −35.106 1.830 −3.724 1.00 41.19 O ATOM 3543 CB SER C 159 −37.190 3.635 −3.343 1.00 41.54 C ATOM 3544 OG SER C 159 −38.272 4.392 −2.822 1.00 47.75 O ATOM 3545 N GLY C 160 −33.693 2.846 −2.281 1.00 38.65 N ATOM 3546 CA GLY C 160 −32.482 2.137 −2.689 1.00 39.01 C ATOM 3547 C GLY C 160 −31.542 2.951 −3.596 1.00 43.01 C ATOM 3548 O GLY C 160 −30.411 2.527 −3.788 1.00 43.49 O ATOM 3549 N ALA C 161 −31.981 4.093 −4.154 1.00 41.35 N ATOM 3550 CA ALA C 161 −31.123 4.921 −5.027 1.00 42.93 C ATOM 3551 C ALA C 161 −29.924 5.544 −4.287 1.00 46.99 C ATOM 3552 O ALA C 161 −28.918 5.833 −4.928 1.00 48.13 O ATOM 3553 CB ALA C 161 −31.942 6.031 −5.687 1.00 44.18 C ATOM 3554 N LEU C 162 −30.031 5.766 −2.957 1.00 41.98 N ATOM 3555 CA LEU C 162 −28.964 6.368 −2.158 1.00 40.66 C ATOM 3556 C LEU C 162 −28.381 5.330 −1.195 1.00 43.86 C ATOM 3557 O LEU C 162 −29.060 4.889 −0.271 1.00 41.50 O ATOM 3558 CB LEU C 162 −29.533 7.567 −1.389 1.00 39.63 C ATOM 3559 CG LEU C 162 −28.580 8.332 −0.469 1.00 42.63 C ATOM 3560 CD1 LEU C 162 −27.376 8.842 −1.225 1.00 43.36 C ATOM 3561 CD2 LEU C 162 −29.306 9.486 0.193 1.00 43.56 C ATOM 3562 N THR C 163 −27.135 4.922 −1.438 1.00 42.81 N ATOM 3563 CA THR C 163 −26.435 3.914 −0.627 1.00 42.79 C ATOM 3564 C THR C 163 −25.122 4.420 −0.023 1.00 48.11 C ATOM 3565 O THR C 163 −24.741 3.965 1.055 1.00 49.40 O ATOM 3566 CB THR C 163 −26.143 2.677 −1.500 1.00 51.93 C ATOM 3567 OG1 THR C 163 −25.384 3.079 −2.650 1.00 54.16 O ATOM 3568 CG2 THR C 163 −27.414 1.975 −1.952 1.00 49.57 C ATOM 3569 N SER C 164 −24.402 5.295 −0.741 1.00 45.94 N ATOM 3570 CA SER C 164 −23.113 5.830 −0.316 1.00 47.16 C ATOM 3571 C SER C 164 −23.266 6.742 0.914 1.00 51.49 C ATOM 3572 O SER C 164 −24.199 7.546 0.974 1.00 50.70 O ATOM 3573 CB SER C 164 −22.465 6.596 −1.470 1.00 52.38 C ATOM 3574 OG SER C 164 −21.112 6.917 −1.197 1.00 64.54 O ATOM 3575 N GLY C 165 −22.380 6.570 1.910 1.00 48.80 N ATOM 3576 CA GLY C 165 −22.378 7.373 3.132 1.00 48.55 C ATOM 3577 C GLY C 165 −23.538 7.086 4.100 1.00 49.65 C ATOM 3578 O GLY C 165 −23.696 7.844 5.054 1.00 50.49 O ATOM 3579 N VAL C 166 −24.329 6.016 3.883 1.00 42.72 N ATOM 3580 CA VAL C 166 −25.470 5.686 4.745 1.00 40.16 C ATOM 3581 C VAL C 166 −25.000 4.874 5.958 1.00 43.26 C ATOM 3582 O VAL C 166 −24.337 3.861 5.769 1.00 43.80 O ATOM 3583 CB VAL C 166 −26.566 4.900 3.966 1.00 42.30 C ATOM 3584 CG1 VAL C 166 −27.690 4.421 4.901 1.00 40.35 C ATOM 3585 CG2 VAL C 166 −27.133 5.731 2.819 1.00 41.87 C ATOM 3586 N HIS C 167 −25.385 5.284 7.184 1.00 38.32 N ATOM 3587 CA HIS C 167 −25.079 4.544 8.404 1.00 37.74 C ATOM 3588 C HIS C 167 −26.377 4.320 9.154 1.00 40.29 C ATOM 3589 O HIS C 167 −26.933 5.278 9.681 1.00 40.72 O ATOM 3590 CB HIS C 167 −24.093 5.295 9.316 1.00 40.12 C ATOM 3591 CG HIS C 167 −22.748 5.542 8.714 1.00 45.66 C ATOM 3592 ND1 HIS C 167 −21.895 4.506 8.392 1.00 47.59 N ATOM 3593 CD2 HIS C 167 −22.144 6.712 8.410 1.00 49.56 C ATOM 3594 CE1 HIS C 167 −20.807 5.065 7.902 1.00 48.72 C ATOM 3595 NE2 HIS C 167 −20.907 6.398 7.901 1.00 50.53 N ATOM 3596 N THR C 168 −26.874 3.077 9.191 1.00 35.88 N ATOM 3597 CA THR C 168 −28.071 2.714 9.963 1.00 35.19 C ATOM 3598 C THR C 168 −27.558 2.063 11.235 1.00 39.47 C ATOM 3599 O THR C 168 −26.951 0.993 11.177 1.00 39.80 O ATOM 3600 CB THR C 168 −29.014 1.829 9.147 1.00 36.19 C ATOM 3601 OG1 THR C 168 −29.437 2.596 8.022 1.00 34.80 O ATOM 3602 CG2 THR C 168 −30.242 1.387 9.939 1.00 31.62 C ATOM 3603 N PHE C 169 −27.768 2.727 12.376 1.00 33.66 N ATOM 3604 CA PHE C 169 −27.248 2.281 13.652 1.00 33.21 C ATOM 3605 C PHE C 169 −27.932 1.027 14.179 1.00 38.49 C ATOM 3606 O PHE C 169 −29.115 0.826 13.902 1.00 36.76 O ATOM 3607 CB PHE C 169 −27.371 3.405 14.708 1.00 34.82 C ATOM 3608 CG PHE C 169 −26.378 4.504 14.457 1.00 37.02 C ATOM 3609 CD1 PHE C 169 −26.692 5.572 13.621 1.00 39.48 C ATOM 3610 CD2 PHE C 169 −25.085 4.406 14.934 1.00 38.66 C ATOM 3611 CE1 PHE C 169 −25.753 6.563 13.347 1.00 41.07 C ATOM 3612 CE2 PHE C 169 −24.145 5.376 14.635 1.00 42.25 C ATOM 3613 CZ PHE C 169 −24.477 6.447 13.835 1.00 40.95 C ATOM 3614 N PRO C 170 −27.220 0.226 15.014 1.00 37.64 N ATOM 3615 CA PRO C 170 −27.876 −0.919 15.637 1.00 36.91 C ATOM 3616 C PRO C 170 −28.952 −0.416 16.590 1.00 39.98 C ATOM 3617 O PRO C 170 −28.785 0.647 17.194 1.00 38.00 O ATOM 3618 CB PRO C 170 −26.741 −1.620 16.398 1.00 39.13 C ATOM 3619 CG PRO C 170 −25.528 −1.261 15.681 1.00 44.32 C ATOM 3620 CD PRO C 170 −25.748 0.119 15.129 1.00 40.17 C ATOM 3621 N ALA C 171 −30.048 −1.172 16.732 1.00 37.84 N ATOM 3622 CA ALA C 171 −31.127 −0.797 17.652 1.00 37.38 C ATOM 3623 C ALA C 171 −30.666 −0.839 19.100 1.00 40.61 C ATOM 3624 O ALA C 171 −29.804 −1.637 19.458 1.00 40.78 O ATOM 3625 CB ALA C 171 −32.308 −1.735 17.483 1.00 37.47 C ATOM 3626 N VAL C 172 −31.237 0.026 19.933 1.00 37.47 N ATOM 3627 CA VAL C 172 −30.945 0.044 21.358 1.00 37.51 C ATOM 3628 C VAL C 172 −32.266 −0.167 22.054 1.00 40.36 C ATOM 3629 O VAL C 172 −33.243 0.510 21.730 1.00 40.15 O ATOM 3630 CB VAL C 172 −30.243 1.343 21.822 1.00 42.01 C ATOM 3631 CG1 VAL C 172 −30.056 1.348 23.337 1.00 43.19 C ATOM 3632 CG2 VAL C 172 −28.893 1.497 21.135 1.00 41.67 C ATOM 3633 N LEU C 173 −32.294 −1.100 23.010 1.00 36.43 N ATOM 3634 CA LEU C 173 −33.475 −1.382 23.802 1.00 36.35 C ATOM 3635 C LEU C 173 −33.518 −0.339 24.929 1.00 44.13 C ATOM 3636 O LEU C 173 −32.555 −0.209 25.697 1.00 44.92 O ATOM 3637 CB LEU C 173 −33.394 −2.817 24.373 1.00 35.69 C ATOM 3638 CG LEU C 173 −34.542 −3.260 25.278 1.00 40.30 C ATOM 3639 CD1 LEU C 173 −35.871 −3.230 24.534 1.00 38.92 C ATOM 3640 CD2 LEU C 173 −34.275 −4.664 25.852 1.00 43.69 C ATOM 3641 N GLN C 174 −34.630 0.404 25.020 1.00 41.93 N ATOM 3642 CA GLN C 174 −34.832 1.442 26.034 1.00 42.66 C ATOM 3643 C GLN C 174 −35.531 0.826 27.250 1.00 46.20 C ATOM 3644 O GLN C 174 −36.099 −0.265 27.133 1.00 42.26 O ATOM 3645 CB GLN C 174 −35.660 2.594 25.433 1.00 44.53 C ATOM 3646 CG GLN C 174 −34.969 3.287 24.229 1.00 51.59 C ATOM 3647 CD GLN C 174 −35.915 3.885 23.210 1.00 62.45 C ATOM 3648 OE1 GLN C 174 −35.838 5.076 22.881 1.00 58.44 O ATOM 3649 NE2 GLN C 174 −36.730 3.052 22.581 1.00 43.24 N ATOM 3650 N SER C 175 −35.503 1.524 28.416 1.00 46.90 N ATOM 3651 CA SER C 175 −36.145 1.046 29.654 1.00 49.01 C ATOM 3652 C SER C 175 −37.661 0.848 29.493 1.00 53.54 C ATOM 3653 O SER C 175 −38.252 0.084 30.256 1.00 54.70 O ATOM 3654 CB SER C 175 −35.850 1.977 30.833 1.00 56.29 C ATOM 3655 OG SER C 175 −35.934 3.339 30.447 1.00 68.49 O ATOM 3656 N SER C 176 −38.276 1.506 28.488 1.00 48.91 N ATOM 3657 CA SER C 176 −39.689 1.336 28.135 1.00 48.23 C ATOM 3658 C SER C 176 −40.004 −0.069 27.584 1.00 49.92 C ATOM 3659 O SER C 176 −41.181 −0.423 27.482 1.00 49.96 O ATOM 3660 CB SER C 176 −40.079 2.345 27.057 1.00 49.39 C ATOM 3661 OG SER C 176 −39.322 2.146 25.871 1.00 50.49 O ATOM 3662 N GLY C 177 −38.975 −0.823 27.143 1.00 44.04 N ATOM 3663 CA GLY C 177 −39.147 −2.140 26.541 1.00 41.89 C ATOM 3664 C GLY C 177 −39.260 −2.024 25.012 1.00 42.93 C ATOM 3665 O GLY C 177 −39.486 −3.036 24.357 1.00 42.28 O ATOM 3666 N LEU C 178 −39.110 −0.809 24.439 1.00 38.86 N ATOM 3667 CA LEU C 178 −39.198 −0.606 22.994 1.00 38.16 C ATOM 3668 C LEU C 178 −37.835 −0.262 22.465 1.00 39.69 C ATOM 3669 O LEU C 178 −37.031 0.363 23.169 1.00 39.65 O ATOM 3670 CB LEU C 178 −40.165 0.529 22.629 1.00 39.12 C ATOM 3671 CG LEU C 178 −41.553 0.412 23.243 1.00 47.25 C ATOM 3672 CD1 LEU C 178 −42.316 1.689 23.084 1.00 49.72 C ATOM 3673 CD2 LEU C 178 −42.333 −0.750 22.645 1.00 48.05 C ATOM 3674 N TYR C 179 −37.606 −0.595 21.197 1.00 32.89 N ATOM 3675 CA TYR C 179 −36.354 −0.311 20.517 1.00 31.68 C ATOM 3676 C TYR C 179 −36.410 1.029 19.789 1.00 34.45 C ATOM 3677 O TYR C 179 −37.491 1.508 19.423 1.00 32.55 O ATOM 3678 CB TYR C 179 −36.048 −1.407 19.493 1.00 31.24 C ATOM 3679 CG TYR C 179 −35.866 −2.771 20.114 1.00 33.01 C ATOM 3680 CD1 TYR C 179 −34.622 −3.194 20.562 1.00 34.09 C ATOM 3681 CD2 TYR C 179 −36.938 −3.651 20.237 1.00 33.66 C ATOM 3682 CE1 TYR C 179 −34.446 −4.459 21.119 1.00 34.49 C ATOM 3683 CE2 TYR C 179 −36.780 −4.908 20.816 1.00 34.17 C ATOM 3684 CZ TYR C 179 −35.532 −5.309 21.256 1.00 39.19 C ATOM 3685 OH TYR C 179 −35.390 −6.551 21.817 1.00 39.50 O ATOM 3686 N SER C 180 −35.224 1.597 19.531 1.00 31.13 N ATOM 3687 CA SER C 180 −35.072 2.814 18.741 1.00 31.06 C ATOM 3688 C SER C 180 −33.736 2.809 18.041 1.00 35.97 C ATOM 3689 O SER C 180 −32.766 2.228 18.541 1.00 35.27 O ATOM 3690 CB SER C 180 −35.166 4.068 19.607 1.00 35.60 C ATOM 3691 OG SER C 180 −36.493 4.283 20.050 1.00 46.10 O ATOM 3692 N LEU C 181 −33.683 3.480 16.897 1.00 33.23 N ATOM 3693 CA LEU C 181 −32.450 3.666 16.146 1.00 33.56 C ATOM 3694 C LEU C 181 −32.552 4.860 15.246 1.00 37.99 C ATOM 3695 O LEU C 181 −33.648 5.366 15.003 1.00 38.13 O ATOM 3696 CB LEU C 181 −32.047 2.415 15.341 1.00 32.57 C ATOM 3697 CG LEU C 181 −32.981 1.912 14.235 1.00 36.26 C ATOM 3698 CD1 LEU C 181 −32.790 2.686 12.914 1.00 37.15 C ATOM 3699 CD2 LEU C 181 −32.694 0.435 13.923 1.00 39.02 C ATOM 3700 N SER C 182 −31.407 5.290 14.719 1.00 35.19 N ATOM 3701 CA SER C 182 −31.337 6.361 13.736 1.00 34.28 C ATOM 3702 C SER C 182 −30.638 5.814 12.503 1.00 35.76 C ATOM 3703 O SER C 182 −29.878 4.852 12.590 1.00 33.49 O ATOM 3704 CB SER C 182 −30.569 7.561 14.283 1.00 36.84 C ATOM 3705 OG SER C 182 −31.440 8.441 14.970 1.00 44.76 O ATOM 3706 N SER C 183 −30.940 6.407 11.354 1.00 33.35 N ATOM 3707 CA SER C 183 −30.297 6.110 10.075 1.00 31.96 C ATOM 3708 C SER C 183 −29.869 7.446 9.541 1.00 35.95 C ATOM 3709 O SER C 183 −30.696 8.351 9.455 1.00 35.40 O ATOM 3710 CB SER C 183 −31.249 5.422 9.107 1.00 32.59 C ATOM 3711 OG SER C 183 −30.574 5.155 7.889 1.00 36.23 O ATOM 3712 N VAL C 184 −28.577 7.612 9.276 1.00 33.92 N ATOM 3713 CA VAL C 184 −28.038 8.886 8.819 1.00 35.09 C ATOM 3714 C VAL C 184 −27.325 8.724 7.483 1.00 40.08 C ATOM 3715 O VAL C 184 −26.886 7.628 7.144 1.00 39.18 O ATOM 3716 CB VAL C 184 −27.109 9.531 9.880 1.00 39.70 C ATOM 3717 CG1 VAL C 184 −27.849 9.751 11.197 1.00 38.39 C ATOM 3718 CG2 VAL C 184 −25.839 8.705 10.098 1.00 40.03 C ATOM 3719 N VAL C 185 −27.187 9.833 6.758 1.00 37.40 N ATOM 3720 CA VAL C 185 −26.466 9.896 5.494 1.00 38.30 C ATOM 3721 C VAL C 185 −25.799 11.270 5.358 1.00 43.11 C ATOM 3722 O VAL C 185 −26.386 12.281 5.759 1.00 41.97 O ATOM 3723 CB VAL C 185 −27.370 9.548 4.274 1.00 41.49 C ATOM 3724 CG1 VAL C 185 −28.521 10.541 4.113 1.00 41.26 C ATOM 3725 CG2 VAL C 185 −26.551 9.450 2.986 1.00 41.92 C ATOM 3726 N THR C 186 −24.559 11.297 4.829 1.00 41.34 N ATOM 3727 CA THR C 186 −23.822 12.538 4.579 1.00 42.71 C ATOM 3728 C THR C 186 −23.867 12.806 3.084 1.00 47.23 C ATOM 3729 O THR C 186 −23.590 11.909 2.287 1.00 46.50 O ATOM 3730 CB THR C 186 −22.407 12.478 5.138 1.00 48.72 C ATOM 3731 OG1 THR C 186 −21.753 11.301 4.669 1.00 48.98 O ATOM 3732 CG2 THR C 186 −22.399 12.500 6.651 1.00 46.29 C ATOM 3733 N VAL C 187 −24.253 14.017 2.703 1.00 45.84 N ATOM 3734 CA VAL C 187 −24.396 14.406 1.298 1.00 46.95 C ATOM 3735 C VAL C 187 −23.729 15.761 1.077 1.00 53.16 C ATOM 3736 O VAL C 187 −23.512 16.485 2.057 1.00 52.77 O ATOM 3737 CB VAL C 187 −25.905 14.433 0.906 1.00 49.45 C ATOM 3738 CG1 VAL C 187 −26.569 13.094 1.232 1.00 47.20 C ATOM 3739 CG2 VAL C 187 −26.659 15.583 1.597 1.00 49.08 C ATOM 3740 N PRO C 188 −23.439 16.140 −0.191 1.00 50.85 N ATOM 3741 CA PRO C 188 −22.881 17.477 −0.416 1.00 52.31 C ATOM 3742 C PRO C 188 −23.950 18.504 −0.032 1.00 55.32 C ATOM 3743 O PRO C 188 −25.117 18.319 −0.372 1.00 53.37 O ATOM 3744 CB PRO C 188 −22.565 17.501 −1.922 1.00 55.35 C ATOM 3745 CG PRO C 188 −22.518 16.080 −2.331 1.00 58.18 C ATOM 3746 CD PRO C 188 −23.491 15.369 −1.450 1.00 51.79 C ATOM 3747 N SER C 189 −23.578 19.540 0.712 1.00 53.46 N ATOM 3748 CA SER C 189 −24.528 20.567 1.139 1.00 53.84 C ATOM 3749 C SER C 189 −25.212 21.269 −0.064 1.00 58.42 C ATOM 3750 O SER C 189 −26.388 21.622 0.036 1.00 56.23 O ATOM 3751 CB SER C 189 −23.849 21.570 2.071 1.00 58.51 C ATOM 3752 OG SER C 189 −22.635 22.048 1.520 1.00 73.56 O ATOM 3753 N SER C 190 −24.515 21.383 −1.217 1.00 57.74 N ATOM 3754 CA SER C 190 −25.083 21.977 −2.441 1.00 59.38 C ATOM 3755 C SER C 190 −26.289 21.165 −2.994 1.00 61.93 C ATOM 3756 O SER C 190 −27.193 21.747 −3.589 1.00 61.60 O ATOM 3757 CB SER C 190 −24.008 22.111 −3.518 1.00 64.69 C ATOM 3758 OG SER C 190 −23.314 20.885 −3.690 1.00 73.17 O ATOM 3759 N SER C 191 −26.326 19.843 −2.750 1.00 57.00 N ATOM 3760 CA SER C 191 −27.443 18.992 −3.188 1.00 54.73 C ATOM 3761 C SER C 191 −28.734 19.180 −2.345 1.00 56.38 C ATOM 3762 O SER C 191 −29.772 18.649 −2.732 1.00 54.22 O ATOM 3763 CB SER C 191 −27.034 17.519 −3.181 1.00 56.06 C ATOM 3764 OG SER C 191 −27.060 16.965 −1.874 1.00 59.55 O ATOM 3765 N LEU C 192 −28.691 19.927 −1.220 1.00 53.23 N ATOM 3766 CA LEU C 192 −29.883 20.138 −0.392 1.00 51.91 C ATOM 3767 C LEU C 192 −30.963 20.981 −1.091 1.00 55.14 C ATOM 3768 O LEU C 192 −32.144 20.791 −0.813 1.00 52.70 O ATOM 3769 CB LEU C 192 −29.505 20.763 0.961 1.00 52.37 C ATOM 3770 CG LEU C 192 −28.583 19.926 1.865 1.00 55.88 C ATOM 3771 CD1 LEU C 192 −28.270 20.674 3.142 1.00 56.85 C ATOM 3772 CD2 LEU C 192 −29.177 18.563 2.166 1.00 54.65 C ATOM 3773 N GLY C 193 −30.574 21.877 −2.011 1.00 54.57 N ATOM 3774 CA GLY C 193 −31.528 22.694 −2.755 1.00 55.72 C ATOM 3775 C GLY C 193 −32.011 22.031 −4.060 1.00 61.36 C ATOM 3776 O GLY C 193 −32.898 22.589 −4.709 1.00 62.19 O ATOM 3777 N THR C 194 −31.407 20.891 −4.473 1.00 58.05 N ATOM 3778 CA THR C 194 −31.758 20.205 −5.733 1.00 57.84 C ATOM 3779 C THR C 194 −32.144 18.715 −5.611 1.00 60.18 C ATOM 3780 O THR C 194 −32.796 18.203 −6.521 1.00 60.53 O ATOM 3781 CB THR C 194 −30.600 20.337 −6.733 1.00 59.06 C ATOM 3782 OG1 THR C 194 −29.519 19.488 −6.339 1.00 57.56 O ATOM 3783 CG2 THR C 194 −30.124 21.771 −6.902 1.00 54.99 C ATOM 3784 N GLN C 195 −31.705 18.009 −4.554 1.00 54.49 N ATOM 3785 CA GLN C 195 −31.974 16.581 −4.374 1.00 52.91 C ATOM 3786 C GLN C 195 −32.915 16.368 −3.192 1.00 54.66 C ATOM 3787 O GLN C 195 −32.746 17.002 −2.155 1.00 53.86 O ATOM 3788 CB GLN C 195 −30.643 15.829 −4.136 1.00 54.19 C ATOM 3789 CG GLN C 195 −30.754 14.304 −4.008 1.00 72.46 C ATOM 3790 CD GLN C 195 −31.323 13.648 −5.239 1.00 86.87 C ATOM 3791 OE1 GLN C 195 −30.937 13.967 −6.364 1.00 91.30 O ATOM 3792 NE2 GLN C 195 −32.240 12.709 −5.068 1.00 71.05 N ATOM 3793 N THR C 196 −33.898 15.476 −3.352 1.00 50.65 N ATOM 3794 CA THR C 196 −34.850 15.126 −2.297 1.00 49.27 C ATOM 3795 C THR C 196 −34.312 13.884 −1.558 1.00 49.76 C ATOM 3796 O THR C 196 −33.700 13.017 −2.190 1.00 47.34 O ATOM 3797 CB THR C 196 −36.247 14.899 −2.901 1.00 61.55 C ATOM 3798 OG1 THR C 196 −36.673 16.111 −3.521 1.00 62.45 O ATOM 3799 CG2 THR C 196 −37.274 14.494 −1.863 1.00 61.37 C ATOM 3800 N TYR C 197 −34.528 13.825 −0.217 1.00 44.93 N ATOM 3801 CA TYR C 197 −34.078 12.725 0.651 1.00 42.40 C ATOM 3802 C TYR C 197 −35.245 12.150 1.432 1.00 45.32 C ATOM 3803 O TYR C 197 −35.835 12.854 2.249 1.00 44.98 O ATOM 3804 CB TYR C 197 −32.975 13.207 1.605 1.00 42.62 C ATOM 3805 CG TYR C 197 −31.729 13.597 0.847 1.00 43.63 C ATOM 3806 CD1 TYR C 197 −30.946 12.634 0.221 1.00 45.06 C ATOM 3807 CD2 TYR C 197 −31.410 14.936 0.632 1.00 44.12 C ATOM 3808 CE1 TYR C 197 −29.886 12.989 −0.609 1.00 47.00 C ATOM 3809 CE2 TYR C 197 −30.313 15.303 −0.147 1.00 45.53 C ATOM 3810 CZ TYR C 197 −29.556 14.324 −0.774 1.00 50.78 C ATOM 3811 OH TYR C 197 −28.449 14.638 −1.528 1.00 47.61 O ATOM 3812 N ILE C 198 −35.606 10.885 1.147 1.00 41.27 N ATOM 3813 CA ILE C 198 −36.717 10.190 1.796 1.00 40.27 C ATOM 3814 C ILE C 198 −36.176 8.897 2.394 1.00 42.85 C ATOM 3815 O ILE C 198 −35.609 8.100 1.658 1.00 43.61 O ATOM 3816 CB ILE C 198 −37.849 9.880 0.759 1.00 43.87 C ATOM 3817 CG1 ILE C 198 −38.453 11.182 0.162 1.00 45.20 C ATOM 3818 CG2 ILE C 198 −38.966 9.018 1.390 1.00 42.53 C ATOM 3819 CD1 ILE C 198 −39.164 10.993 −1.177 1.00 47.15 C ATOM 3820 N CYS C 199 −36.385 8.655 3.694 1.00 38.95 N ATOM 3821 CA CYS C 199 −35.992 7.381 4.304 1.00 37.58 C ATOM 3822 C CYS C 199 −37.198 6.464 4.219 1.00 37.49 C ATOM 3823 O CYS C 199 −38.322 6.921 4.408 1.00 36.74 O ATOM 3824 CB CYS C 199 −35.504 7.552 5.744 1.00 38.53 C ATOM 3825 SG CYS C 199 −36.774 8.077 6.929 1.00 43.26 S ATOM 3826 N ASN C 200 −36.978 5.195 3.891 1.00 33.95 N ATOM 3827 CA ASN C 200 −38.060 4.216 3.760 1.00 34.51 C ATOM 3828 C ASN C 200 −37.888 3.245 4.910 1.00 36.82 C ATOM 3829 O ASN C 200 −36.941 2.470 4.916 1.00 36.20 O ATOM 3830 CB ASN C 200 −38.011 3.500 2.405 1.00 37.20 C ATOM 3831 CG ASN C 200 −37.611 4.407 1.280 1.00 44.47 C ATOM 3832 OD1 ASN C 200 −36.480 4.369 0.806 1.00 38.46 O ATOM 3833 ND2 ASN C 200 −38.482 5.318 0.910 1.00 34.99 N ATOM 3834 N VAL C 201 −38.753 3.340 5.912 1.00 33.44 N ATOM 3835 CA VAL C 201 −38.680 2.509 7.115 1.00 32.43 C ATOM 3836 C VAL C 201 −39.695 1.366 7.027 1.00 36.27 C ATOM 3837 O VAL C 201 −40.873 1.619 6.803 1.00 36.91 O ATOM 3838 CB VAL C 201 −38.907 3.383 8.367 1.00 34.96 C ATOM 3839 CG1 VAL C 201 −38.753 2.562 9.656 1.00 34.55 C ATOM 3840 CG2 VAL C 201 −37.945 4.568 8.366 1.00 34.28 C ATOM 3841 N ASN C 202 −39.250 0.122 7.210 1.00 32.13 N ATOM 3842 CA ASN C 202 −40.152 −1.027 7.170 1.00 32.50 C ATOM 3843 C ASN C 202 −40.048 −1.791 8.481 1.00 35.35 C ATOM 3844 O ASN C 202 −38.947 −2.073 8.959 1.00 34.29 O ATOM 3845 CB ASN C 202 −39.827 −1.952 5.985 1.00 35.75 C ATOM 3846 CG ASN C 202 −40.905 −2.964 5.659 1.00 69.46 C ATOM 3847 OD1 ASN C 202 −42.031 −2.909 6.159 1.00 68.86 O ATOM 3848 ND2 ASN C 202 −40.578 −3.943 4.838 1.00 63.99 N ATOM 3849 N HIS C 203 −41.192 −2.111 9.068 1.00 32.54 N ATOM 3850 CA HIS C 203 −41.253 −2.904 10.295 1.00 31.61 C ATOM 3851 C HIS C 203 −42.271 −4.017 10.026 1.00 36.97 C ATOM 3852 O HIS C 203 −43.465 −3.845 10.254 1.00 38.03 O ATOM 3853 CB HIS C 203 −41.617 −2.018 11.491 1.00 32.05 C ATOM 3854 CG HIS C 203 −41.663 −2.748 12.799 1.00 34.54 C ATOM 3855 ND1 HIS C 203 −42.846 −2.909 13.479 1.00 36.65 N ATOM 3856 CD2 HIS C 203 −40.673 −3.351 13.497 1.00 34.43 C ATOM 3857 CE1 HIS C 203 −42.550 −3.592 14.571 1.00 35.40 C ATOM 3858 NE2 HIS C 203 −41.253 −3.884 14.621 1.00 34.84 N ATOM 3859 N LYS C 204 −41.786 −5.134 9.453 1.00 33.51 N ATOM 3860 CA LYS C 204 −42.628 −6.274 9.090 1.00 35.51 C ATOM 3861 C LYS C 204 −43.444 −6.849 10.260 1.00 39.45 C ATOM 3862 O LYS C 204 −44.613 −7.156 10.033 1.00 39.43 O ATOM 3863 CB LYS C 204 −41.808 −7.392 8.411 1.00 37.95 C ATOM 3864 CG LYS C 204 −41.383 −7.055 6.983 1.00 59.99 C ATOM 3865 CD LYS C 204 −40.254 −7.979 6.488 1.00 75.65 C ATOM 3866 CE LYS C 204 −40.468 −8.523 5.093 1.00 89.72 C ATOM 3867 NZ LYS C 204 −40.459 −7.456 4.063 1.00 98.79 N ATOM 3868 N PRO C 205 −42.889 −6.924 11.511 1.00 34.46 N ATOM 3869 CA PRO C 205 −43.669 −7.486 12.626 1.00 34.26 C ATOM 3870 C PRO C 205 −45.016 −6.787 12.912 1.00 40.22 C ATOM 3871 O PRO C 205 −45.938 −7.472 13.350 1.00 42.16 O ATOM 3872 CB PRO C 205 −42.698 −7.408 13.801 1.00 34.66 C ATOM 3873 CG PRO C 205 −41.356 −7.530 13.172 1.00 37.43 C ATOM 3874 CD PRO C 205 −41.487 −6.694 11.929 1.00 33.27 C ATOM 3875 N SER C 206 −45.151 −5.471 12.609 1.00 36.17 N ATOM 3876 CA SER C 206 −46.398 −4.713 12.784 1.00 37.09 C ATOM 3877 C SER C 206 −47.056 −4.313 11.446 1.00 45.52 C ATOM 3878 O SER C 206 −48.057 −3.587 11.460 1.00 46.22 O ATOM 3879 CB SER C 206 −46.133 −3.451 13.600 1.00 37.24 C ATOM 3880 OG SER C 206 −45.481 −2.468 12.815 1.00 39.38 O ATOM 3881 N ASN C 207 −46.501 −4.765 10.305 1.00 44.74 N ATOM 3882 CA ASN C 207 −46.968 −4.420 8.959 1.00 46.37 C ATOM 3883 C ASN C 207 −46.954 −2.893 8.750 1.00 51.08 C ATOM 3884 O ASN C 207 −47.877 −2.330 8.164 1.00 52.75 O ATOM 3885 CB ASN C 207 −48.348 −5.039 8.668 1.00 51.57 C ATOM 3886 CG ASN C 207 −48.734 −5.047 7.199 1.00 84.75 C ATOM 3887 OD1 ASN C 207 −47.888 −5.160 6.298 1.00 80.06 O ATOM 3888 ND2 ASN C 207 −50.024 −4.920 6.913 1.00 79.89 N ATOM 3889 N THR C 208 −45.895 −2.229 9.243 1.00 46.28 N ATOM 3890 CA THR C 208 −45.734 −0.776 9.148 1.00 45.98 C ATOM 3891 C THR C 208 −44.659 −0.441 8.109 1.00 49.44 C ATOM 3892 O THR C 208 −43.551 −0.980 8.176 1.00 48.86 O ATOM 3893 CB THR C 208 −45.379 −0.203 10.537 1.00 51.02 C ATOM 3894 OG1 THR C 208 −46.494 −0.374 11.410 1.00 50.03 O ATOM 3895 CG2 THR C 208 −44.994 1.271 10.495 1.00 52.32 C ATOM 3896 N LYS C 209 −45.007 0.417 7.140 1.00 45.59 N ATOM 3897 CA LYS C 209 −44.097 0.942 6.119 1.00 44.94 C ATOM 3898 C LYS C 209 −44.286 2.450 6.160 1.00 48.08 C ATOM 3899 O LYS C 209 −45.421 2.906 6.027 1.00 49.70 O ATOM 3900 CB LYS C 209 −44.415 0.401 4.708 1.00 47.52 C ATOM 3901 CG LYS C 209 −43.607 −0.845 4.335 1.00 60.67 C ATOM 3902 CD LYS C 209 −43.547 −1.105 2.822 1.00 68.70 C ATOM 3903 CE LYS C 209 −44.819 −1.659 2.229 1.00 79.44 C ATOM 3904 NZ LYS C 209 −44.942 −3.128 2.426 1.00 90.37 N ATOM 3905 N VAL C 210 −43.209 3.214 6.404 1.00 42.04 N ATOM 3906 CA VAL C 210 −43.271 4.679 6.496 1.00 41.81 C ATOM 3907 C VAL C 210 −42.211 5.272 5.578 1.00 45.20 C ATOM 3908 O VAL C 210 −41.067 4.830 5.608 1.00 44.94 O ATOM 3909 CB VAL C 210 −43.045 5.153 7.961 1.00 45.14 C ATOM 3910 CG1 VAL C 210 −43.168 6.675 8.080 1.00 45.33 C ATOM 3911 CG2 VAL C 210 −44.010 4.459 8.922 1.00 45.25 C ATOM 3912 N ASP C 211 −42.588 6.271 4.778 1.00 42.34 N ATOM 3913 CA ASP C 211 −41.678 7.018 3.919 1.00 41.90 C ATOM 3914 C ASP C 211 −41.670 8.429 4.488 1.00 47.72 C ATOM 3915 O ASP C 211 −42.726 9.066 4.503 1.00 50.20 O ATOM 3916 CB ASP C 211 −42.175 7.056 2.465 1.00 43.60 C ATOM 3917 CG ASP C 211 −42.166 5.711 1.779 1.00 52.70 C ATOM 3918 OD1 ASP C 211 −41.161 4.981 1.914 1.00 51.11 O ATOM 3919 OD2 ASP C 211 −43.156 5.390 1.096 1.00 63.38 O ATOM 3920 N LYS C 212 −40.526 8.908 4.996 1.00 41.79 N ATOM 3921 CA LYS C 212 −40.451 10.257 5.554 1.00 41.69 C ATOM 3922 C LYS C 212 −39.464 11.107 4.774 1.00 44.76 C ATOM 3923 O LYS C 212 −38.279 10.792 4.736 1.00 43.28 O ATOM 3924 CB LYS C 212 −40.092 10.237 7.053 1.00 42.89 C ATOM 3925 CG LYS C 212 −40.062 11.641 7.694 1.00 52.40 C ATOM 3926 CD LYS C 212 −41.179 11.885 8.695 1.00 64.22 C ATOM 3927 CE LYS C 212 −42.575 11.953 8.125 1.00 78.55 C ATOM 3928 NZ LYS C 212 −43.444 10.866 8.657 1.00 90.01 N ATOM 3929 N LYS C 213 −39.953 12.210 4.195 1.00 43.38 N ATOM 3930 CA LYS C 213 −39.131 13.173 3.463 1.00 44.27 C ATOM 3931 C LYS C 213 −38.375 14.004 4.506 1.00 47.22 C ATOM 3932 O LYS C 213 −38.991 14.502 5.444 1.00 47.71 O ATOM 3933 CB LYS C 213 −40.033 14.070 2.588 1.00 48.79 C ATOM 3934 CG LYS C 213 −39.311 14.910 1.543 1.00 67.33 C ATOM 3935 CD LYS C 213 −40.324 15.788 0.808 1.00 83.39 C ATOM 3936 CE LYS C 213 −39.715 16.704 −0.225 1.00 99.34 C ATOM 3937 NZ LYS C 213 −40.684 17.733 −0.697 1.00 109.18 N ATOM 3938 N VAL C 214 −37.040 14.082 4.391 1.00 43.34 N ATOM 3939 CA VAL C 214 −36.184 14.827 5.318 1.00 42.97 C ATOM 3940 C VAL C 214 −35.825 16.116 4.604 1.00 52.25 C ATOM 3941 O VAL C 214 −35.042 16.086 3.660 1.00 52.40 O ATOM 3942 CB VAL C 214 −34.937 14.003 5.716 1.00 44.58 C ATOM 3943 CG1 VAL C 214 −34.052 14.771 6.695 1.00 43.89 C ATOM 3944 CG2 VAL C 214 −35.356 12.662 6.314 1.00 43.24 C ATOM 3945 N GLU C 215 −36.417 17.238 5.034 1.00 53.37 N ATOM 3946 CA GLU C 215 −36.239 18.544 4.391 1.00 55.97 C ATOM 3947 C GLU C 215 −35.388 19.506 5.228 1.00 62.07 C ATOM 3948 O GLU C 215 −35.449 19.455 6.456 1.00 59.01 O ATOM 3949 CB GLU C 215 −37.621 19.180 4.165 1.00 57.99 C ATOM 3950 CG GLU C 215 −38.511 18.370 3.237 1.00 69.95 C ATOM 3951 CD GLU C 215 −39.907 18.931 3.064 1.00 99.06 C ATOM 3952 OE1 GLU C 215 −40.554 19.249 4.088 1.00 96.25 O ATOM 3953 OE2 GLU C 215 −40.369 19.021 1.904 1.00 100.10 O ATOM 3954 N PRO C 216 −34.630 20.421 4.570 1.00 64.54 N ATOM 3955 CA PRO C 216 −33.890 21.440 5.317 1.00 67.05 C ATOM 3956 C PRO C 216 −34.951 22.418 5.830 1.00 73.58 C ATOM 3957 O PRO C 216 −35.415 23.272 5.075 1.00 75.08 O ATOM 3958 CB PRO C 216 −32.955 22.057 4.267 1.00 69.95 C ATOM 3959 CG PRO C 216 −33.618 21.819 2.976 1.00 73.76 C ATOM 3960 CD PRO C 216 −34.516 20.629 3.111 1.00 67.15 C ATOM 3961 N LYS C 217 −35.421 22.196 7.062 1.00 71.52 N ATOM 3962 CA LYS C 217 −36.490 22.981 7.683 1.00 73.15 C ATOM 3963 C LYS C 217 −35.916 24.214 8.389 1.00 77.06 C ATOM 3964 O LYS C 217 −35.811 25.276 7.778 1.00 76.66 O ATOM 3965 CB LYS C 217 −37.283 22.094 8.671 1.00 76.19 C ATOM 3966 CG LYS C 217 −38.705 22.587 8.990 1.00 97.50 C ATOM 3967 CD LYS C 217 −38.758 23.599 10.147 1.00 107.97 C ATOM 3968 CE LYS C 217 −40.162 23.851 10.650 1.00 117.10 C ATOM 3969 NZ LYS C 217 −40.963 24.677 9.709 1.00 125.01 N ATOM 3970 CD CD C 9901 −22.410 2.405 7.030 0.70 74.10 CD ATOM 3971 CD CD C 9902 −42.496 −21.639 17.273 0.70 68.69 CD ATOM 3972 OW WAT W 1 −43.686 6.507 20.633 1.00 37.64 O ATOM 3973 OW WAT W 2 −7.515 −55.973 40.942 1.00 34.99 O ATOM 3974 OW WAT W 3 −16.190 −32.676 18.735 1.00 49.40 O ATOM 3975 OW WAT W 4 −17.557 −57.264 27.490 1.00 49.31 O ATOM 3976 OW WAT W 5 −6.453 −52.025 23.346 1.00 34.24 O ATOM 3977 OW WAT W 6 −4.793 −51.902 29.479 1.00 34.78 O ATOM 3978 OW WAT W 7 1.021 −54.897 36.399 1.00 30.59 O ATOM 3979 OW WAT W 8 −22.290 −23.802 30.481 1.00 54.43 O ATOM 3980 OW WAT W 9 −29.894 19.889 12.291 1.00 45.45 O ATOM 3981 OW WAT W 10 −4.843 −65.658 37.110 1.00 52.73 O ATOM 3982 OW WAT W 11 −13.185 −6.989 13.786 1.00 52.53 O ATOM 3983 OW WAT W 12 −29.240 0.950 5.923 1.00 42.90 O ATOM 3984 OW WAT W 13 −7.340 −61.094 23.749 1.00 48.50 O ATOM 3985 OW WAT W 14 −43.809 −9.689 23.593 1.00 40.85 O ATOM 3986 OW WAT W 15 −36.983 6.868 25.062 1.00 43.15 O ATOM 3987 OW WAT W 16 −19.890 −39.199 26.687 1.00 59.47 O ATOM 3988 OW WAT W 17 −47.034 −12.817 30.547 1.00 68.03 O ATOM 3989 OW WAT W 18 −8.001 1.396 20.051 1.00 52.96 O ATOM 3990 OW WAT W 19 −38.870 4.456 24.555 1.00 38.93 O ATOM 3991 OW WAT W 20 −22.038 −2.265 17.233 1.00 64.74 O ATOM 3992 OW WAT W 21 −4.829 −23.889 19.435 1.00 55.04 O ATOM 3993 OW WAT W 22 −13.553 −58.794 35.421 1.00 47.74 O ATOM 3994 OW WAT W 23 3.472 −56.247 37.587 1.00 37.92 O ATOM 3995 OW WAT W 24 −9.774 −61.094 20.609 1.00 55.83 O ATOM 3996 OW WAT W 25 −1.424 −58.706 31.053 1.00 34.00 O ATOM 3997 OW WAT W 26 −14.822 −20.200 29.926 1.00 51.09 O ATOM 3998 OW WAT W 27 −48.117 −0.157 15.298 1.00 50.50 O ATOM 3999 OW WAT W 28 −18.464 −54.748 39.058 1.00 49.62 O ATOM 4000 OW WAT W 29 −16.944 14.679 26.129 1.00 61.00 O ATOM 4001 OW WAT W 30 −38.364 16.745 35.129 1.00 41.91 O ATOM 4002 OW WAT W 31 −15.959 −35.971 25.563 1.00 59.27 O ATOM 4003 OW WAT W 32 −12.962 −32.423 10.242 1.00 47.37 O ATOM 4004 OW WAT W 33 −17.151 −34.677 22.424 1.00 41.49 O ATOM 4005 OW WAT W 34 −39.535 2.389 −1.048 1.00 52.38 O ATOM 4006 OW WAT W 35 −16.188 −53.762 40.168 1.00 36.04 O ATOM 4007 OW WAT W 36 −15.801 −27.934 32.197 1.00 51.21 O ATOM 4008 OW WAT W 37 −42.619 12.844 4.552 1.00 48.29 O ATOM 4009 OW WAT W 38 −40.075 −7.299 29.641 1.00 45.60 O ATOM 4010 OW WAT W 39 −25.307 −43.940 25.007 1.00 46.30 O ATOM 4011 OW WAT W 40 −13.473 −59.058 32.594 1.00 39.53 O ATOM 4012 OW WAT W 41 −13.787 −45.903 30.958 1.00 41.44 O ATOM 4013 OW WAT W 42 −1.167 −62.434 35.598 1.00 45.52 O ATOM 4014 OW WAT W 43 −8.824 13.349 11.279 1.00 60.80 O ATOM 4015 OW WAT W 44 −9.014 −54.295 21.103 1.00 48.13 O ATOM 4016 OW WAT W 45 −33.709 −17.937 9.821 1.00 47.87 O ATOM 4017 OW WAT W 46 −42.571 23.661 29.793 1.00 53.44 O ATOM 4018 OW WAT W 47 −23.013 −36.129 28.963 1.00 57.82 O ATOM 4019 OW WAT W 48 −39.976 −30.578 24.711 1.00 49.01 O ATOM 4020 OW WAT W 49 −12.230 −37.480 14.745 1.00 52.37 O ATOM 4021 OW WAT W 50 −40.880 −7.265 1.223 1.00 46.91 O ATOM 4022 OW WAT W 51 −36.217 −8.573 26.196 1.00 45.73 O ATOM 4023 OW WAT W 52 −27.369 −42.522 26.037 1.00 50.82 O ATOM 4024 OW WAT W 53 −30.085 −1.525 26.270 1.00 46.62 O ATOM 4025 OW WAT W 54 −21.459 −42.917 23.254 1.00 56.77 O ATOM 4026 OW WAT W 55 −48.128 −9.341 16.581 1.00 52.04 O ATOM 4027 OW WAT W 56 −41.161 2.175 3.473 1.00 53.80 O ATOM 4028 OW WAT W 57 −32.959 −33.970 6.634 1.00 57.70 O ATOM 4029 OW WAT W 58 −22.679 −27.983 34.019 1.00 57.71 O ATOM 4030 OW WAT W 59 −35.865 −35.113 26.946 1.00 52.15 O ATOM 4031 OW WAT W 60 −38.088 17.218 7.521 1.00 43.82 O ATOM 4032 OW WAT W 61 −17.695 −4.736 10.101 1.00 59.45 O ATOM 4033 OW WAT W 62 −29.006 23.048 5.525 1.00 54.53 O ATOM 4034 OW WAT W 63 −2.632 −61.249 33.270 1.00 49.17 O ATOM 4035 OW WAT W 64 −30.677 9.217 −4.428 1.00 46.60 O ATOM 4036 OW WAT W 65 −2.089 −24.886 12.106 1.00 63.36 O ATOM 4037 OW WAT W 66 −46.157 −15.581 24.252 1.00 46.20 O ATOM 4038 OW WAT W 67 −40.525 −32.892 20.549 1.00 55.06 O ATOM 4039 OW WAT W 68 −41.514 −13.927 14.806 1.00 62.38 O ATOM 4040 OW WAT W 69 −22.302 −40.343 25.447 1.00 53.17 O ATOM 4041 OW WAT W 70 −22.643 −30.586 32.221 1.00 47.03 O ATOM 4042 OW WAT W 71 −41.750 −11.218 15.132 1.00 42.49 O ATOM 4043 OW WAT W 72 −16.498 −38.238 11.084 1.00 59.10 O ATOM 4044 OW WAT W 73 −32.508 6.114 19.320 1.00 50.45 O ATOM 4045 OW WAT W 74 −47.798 −8.546 21.176 1.00 54.03 O ATOM 4046 OW WAT W 75 5.501 −62.333 33.875 1.00 40.22 O ATOM 4047 OW WAT W 76 −0.630 −56.746 51.323 1.00 58.43 O ATOM 4048 OW WAT W 77 −14.253 −16.896 30.328 1.00 64.63 O ATOM 4049 OW WAT W 78 −16.129 −44.953 29.427 1.00 44.23 O ATOM 4050 OW WAT W 79 −44.049 14.182 28.247 1.00 40.45 O ATOM 4051 OW WAT W 80 −30.357 −0.086 −3.432 1.00 43.64 O ATOM 4052 OW WAT W 81 −17.609 −55.150 36.210 1.00 47.81 O ATOM 4053 OW WAT W 82 −5.620 −0.309 7.737 1.00 64.98 O ATOM 4054 OW WAT W 83 −35.333 −36.792 24.625 1.00 55.50 O ATOM 4055 OW WAT W 84 −3.864 −28.110 9.290 1.00 58.03 O ATOM 4056 OW WAT W 85 −22.868 −47.176 23.771 1.00 57.37 O ATOM 4057 OW WAT W 86 0.090 −50.852 36.108 1.00 45.20 O ATOM 4058 OW WAT W 87 −8.183 −1.151 21.020 1.00 62.51 O ATOM 4059 OW WAT W 88 −33.018 −7.725 22.304 1.00 45.47 O ATOM 4060 OW WAT W 89 −31.655 −1.393 7.537 1.00 42.27 O ATOM 4061 OW WAT W 90 −32.662 17.636 2.924 1.00 46.29 O ATOM 4062 OW WAT W 91 −17.465 −38.109 26.276 1.00 56.12 O ATOM 4063 OW WAT W 92 −6.887 −60.668 45.840 1.00 56.69 O ATOM 4064 OW WAT W 93 −39.318 −11.867 32.764 1.00 53.22 O ATOM 4065 OW WAT W 94 −8.364 −47.344 15.912 1.00 52.70 O ATOM 4066 OW WAT W 95 −13.110 −2.409 20.213 1.00 59.42 O ATOM 4067 OW WAT W 96 −30.405 −3.733 15.635 1.00 43.90 O ATOM 4068 OW WAT W 97 2.111 −65.266 36.755 1.00 50.54 O ATOM 4069 OW WAT W 98 −12.718 −10.231 25.834 1.00 56.71 O ATOM 4070 OW WAT W 99 −20.180 −52.382 20.505 1.00 56.19 O ATOM 4071 OW WAT W 100 −13.218 −54.275 21.382 1.00 65.09 O ATOM 4072 OW WAT W 101 −0.687 −48.919 38.318 1.00 48.28 O ATOM 4073 OW WAT W 102 −3.838 −62.253 43.700 1.00 49.16 O ATOM 4074 OW WAT W 103 −39.718 4.768 29.789 1.00 49.94 O ATOM 4075 OW WAT W 104 −31.944 −3.089 12.979 1.00 46.48 O ATOM 4076 OW WAT W 105 −33.765 −18.248 12.740 1.00 32.36 O ATOM 4077 OW WAT W 106 −37.214 −0.117 4.446 1.00 29.24 O ATOM 4078 OW WAT W 107 −19.351 −15.874 11.194 1.00 36.71 O ATOM 4079 OW WAT W 108 −21.775 −21.417 10.614 1.00 39.48 O ATOM 4080 OW WAT W 109 −26.619 −35.038 14.715 1.00 32.72 O ATOM 4081 OW WAT W 110 −35.620 13.559 15.135 1.00 36.10 O ATOM 4082 OW WAT W 111 −36.996 −10.846 16.382 1.00 44.68 O ATOM 4083 OW WAT W 112 −36.912 −6.950 24.030 1.00 33.68 O ATOM 4084 OW WAT W 113 −33.516 14.817 16.865 1.00 35.25 O ATOM 4085 OW WAT W 114 −29.783 −1.424 12.325 1.00 39.43 O ATOM 4086 OW WAT W 115 −18.166 −16.800 25.381 1.00 43.01 O ATOM 4087 OW WAT W 116 −24.077 10.140 −0.068 1.00 45.52 O ATOM 4088 OW WAT W 117 −24.361 −0.261 19.500 1.00 42.65 O ATOM 4089 OW WAT W 118 −37.730 14.775 30.946 1.00 36.06 O ATOM 4090 OW WAT W 119 −20.173 −22.700 28.237 1.00 47.60 O ATOM 4091 OW WAT W 120 −24.540 20.221 13.439 1.00 39.18 O ATOM 4092 OW WAT W 121 −17.942 4.938 9.763 1.00 42.28 O ATOM 4093 OW WAT W 122 −38.874 −5.521 9.526 1.00 31.42 O ATOM 4094 OW WAT W 123 −16.780 −29.944 20.969 1.00 38.26 O ATOM 4095 OW WAT W 124 −24.266 −15.743 26.088 1.00 44.60 O ATOM 4096 OW WAT W 125 −29.109 5.403 16.817 1.00 36.92 O ATOM 4097 OW WAT W 126 −26.521 −36.987 26.430 1.00 48.99 O ATOM 4098 OW WAT W 127 −33.883 −9.961 20.793 1.00 33.74 O ATOM 4099 OW WAT W 128 −32.376 −31.327 21.059 1.00 37.71 O ATOM 4100 OW WAT W 129 −38.219 −21.030 16.400 1.00 32.53 O ATOM 4101 OW WAT W 130 −38.266 7.048 −1.804 1.00 26.87 O ATOM 4102 OW WAT W 131 −40.459 8.361 23.021 1.00 36.24 O ATOM 4103 OW WAT W 132 −21.073 −36.674 18.501 1.00 36.84 O ATOM 4104 OW WAT W 133 −36.873 −8.310 19.156 1.00 44.97 O ATOM 4105 OW WAT W 134 −31.678 −18.920 28.463 1.00 36.27 O ATOM 4106 OW WAT W 135 −21.806 −18.434 9.776 1.00 43.63 O ATOM 4107 OW WAT W 136 −46.153 −9.807 14.720 1.00 51.20 O ATOM 4108 OW WAT W 137 −24.475 −44.424 16.285 1.00 47.32 O ATOM 4109 OW WAT W 138 −37.829 −30.269 23.129 1.00 40.12 O ATOM 4110 OW WAT W 139 −29.743 3.061 18.200 1.00 39.38 O ATOM 4111 OW WAT W 140 −26.353 −16.935 10.862 1.00 38.95 O ATOM 4112 OW WAT W 141 −27.059 12.054 29.756 1.00 51.89 O ATOM 4113 OW WAT W 142 −25.585 6.342 −3.334 1.00 43.68 O ATOM 4114 OW WAT W 143 −29.744 −2.526 23.600 1.00 38.01 O ATOM 4115 OW WAT W 144 −28.349 6.767 20.043 1.00 31.75 O ATOM 4116 OW WAT W 145 −10.172 −29.271 24.638 1.00 50.15 O ATOM 4117 OW WAT W 146 −21.497 −32.672 22.980 1.00 43.68 O ATOM 4118 OW WAT W 147 −45.840 −1.515 22.524 1.00 38.43 O ATOM 4119 OW WAT W 148 −30.947 7.489 17.609 1.00 35.91 O ATOM 4120 OW WAT W 149 −35.715 16.046 1.011 1.00 43.17 O ATOM 4121 OW WAT W 150 −34.097 6.759 21.605 1.00 32.44 O ATOM 4122 OW WAT W 151 −31.153 −29.424 6.931 1.00 52.76 O ATOM 4123 OW WAT W 152 −17.488 −27.746 8.560 1.00 51.05 O ATOM 4124 OW WAT W 153 −13.184 −8.413 8.214 1.00 52.08 O ATOM 4125 OW WAT W 154 −9.649 −15.709 6.299 1.00 50.09 O ATOM 4126 OW WAT W 155 −30.314 4.934 20.366 1.00 39.36 O ATOM 4127 OW WAT W 156 −32.747 18.271 0.227 1.00 42.14 O ATOM 4128 OW WAT W 157 −24.802 −10.885 20.391 1.00 46.19 O ATOM 4129 OW WAT W 158 −18.677 −24.593 10.969 1.00 35.79 O ATOM 4130 OW WAT W 159 −25.286 −39.676 26.160 1.00 44.70 O ATOM 4131 OW WAT W 160 −25.878 23.640 27.286 1.00 51.31 O ATOM 4132 OW WAT W 161 −40.111 −30.248 12.723 1.00 46.12 O ATOM 4133 OW WAT W 162 −48.933 11.765 22.678 1.00 39.67 O ATOM 4134 OW WAT W 163 −43.012 −16.049 18.180 1.00 46.36 O ATOM 4135 OW WAT W 164 −24.923 −25.595 8.909 1.00 38.70 O ATOM 4136 OW WAT W 165 −35.301 −33.872 29.546 1.00 51.23 O ATOM 4137 OW WAT W 166 −23.108 −1.358 11.523 1.00 49.25 O ATOM 4138 OW WAT W 167 −21.256 20.978 −1.257 1.00 50.39 O ATOM 4139 OW WAT W 168 −20.045 −30.634 4.149 1.00 58.57 O ATOM 4140 OW WAT W 169 −18.550 −10.595 15.754 1.00 61.81 O ATOM 4141 OW WAT W 170 −18.747 12.952 7.864 1.00 42.13 O ATOM 4142 OW WAT W 171 −24.751 11.499 −2.308 1.00 45.78 O ATOM 4143 OW WAT W 172 −22.547 −39.523 8.065 1.00 46.22 O ATOM 4144 OW WAT W 173 −30.806 2.957 0.490 1.00 40.34 O ATOM 4145 OW WAT W 174 −15.757 −32.291 9.949 1.00 47.94 O ATOM 4146 OW WAT W 175 −44.573 16.854 28.331 1.00 52.33 O ATOM 4147 OW WAT W 176 −7.977 −15.083 21.992 1.00 53.44 O ATOM 4148 OW WAT W 177 −25.074 9.003 −3.979 1.00 50.70 O ATOM 4149 OW WAT W 178 −10.590 0.899 18.154 1.00 47.60 O ATOM 4150 OW WAT W 179 −7.736 −52.251 45.643 1.00 45.32 O ATOM 4151 OW WAT W 180 −44.390 −9.173 18.853 1.00 43.31 O ATOM 4152 OW WAT W 181 −36.893 −15.564 11.325 1.00 40.98 O ATOM 4153 OW WAT W 182 −18.668 −1.614 16.801 1.00 53.28 O ATOM 4154 OW WAT W 183 −36.728 −37.278 22.169 1.00 51.45 O ATOM 4155 OW WAT W 184 −4.056 −59.087 45.469 1.00 47.73 O ATOM 4156 OW WAT W 185 −34.977 17.331 12.959 1.00 50.25 O ATOM 4157 OW WAT W 186 −15.254 −15.990 6.954 1.00 53.33 O ATOM 4158 OW WAT W 187 −25.008 −19.043 9.149 1.00 50.15 O ATOM 4159 OW WAT W 188 −12.479 −14.878 6.876 1.00 58.96 O ATOM 4160 OW WAT W 189 −21.761 −15.537 27.605 1.00 53.73 O ATOM 4161 OW WAT W 190 −13.699 −32.590 19.546 1.00 46.79 O ATOM 4162 OW WAT W 191 −1.653 −67.263 38.821 1.00 49.26 O ATOM 4163 OW WAT W 192 −16.337 −34.412 32.445 1.00 55.13 O ATOM 4164 OW WAT W 193 −40.988 10.137 21.044 1.00 38.32 O ATOM 4165 OW WAT W 194 −8.330 −30.065 33.366 1.00 51.30 O ATOM 4166 OW WAT W 195 −35.850 −26.313 11.313 1.00 45.94 O ATOM 4167 OW WAT W 196 −44.254 −21.966 29.984 1.00 48.92 O ATOM 4168 OW WAT W 197 −14.556 −52.288 19.703 1.00 47.54 O ATOM 4169 OW WAT W 198 −45.543 6.721 4.393 1.00 56.24 O ATOM 4170 OW WAT W 199 −1.510 −52.502 46.012 1.00 49.88 O ATOM 4171 OW WAT W 200 −37.221 −8.769 3.755 1.00 48.71 O ATOM 4172 OW WAT W 201 −15.008 −9.692 6.521 1.00 54.03 O ATOM 4173 OW WAT W 202 −9.154 −10.150 2.409 1.00 64.21 O ATOM 4174 OW WAT W 203 −39.318 −31.340 8.710 1.00 57.51 O ATOM 4175 OW WAT W 204 −45.582 −10.139 21.126 1.00 51.87 O ATOM 4176 OW WAT W 205 −44.026 −10.354 16.431 1.00 43.73 O ATOM 4177 OW WAT W 206 −36.210 7.117 −5.687 1.00 45.43 O ATOM 4178 OW WAT W 207 −37.799 14.409 16.431 1.00 36.95 O ATOM 4179 OW WAT W 208 −34.076 −3.333 14.608 1.00 39.15 O ATOM 4180 OW WAT W 209 −21.963 20.042 12.700 1.00 49.97 O ATOM 4181 OW WAT W 210 −38.664 −5.379 25.427 1.00 34.19 O ATOM 4182 OW WAT W 211 −27.013 −1.595 20.048 1.00 59.63 O ATOM 4183 OW WAT W 212 −34.179 −15.986 14.105 1.00 32.00 O ATOM 4184 OW WAT W 213 −24.410 −21.562 10.160 1.00 36.16 O ATOM 4185 OW WAT W 214 −39.138 −10.516 14.813 1.00 47.91 O ATOM 4186 OW WAT W 215 −38.147 −4.980 6.998 1.00 40.50 O ATOM 4187 OW WAT W 216 −21.263 −24.053 10.299 1.00 37.28 O ATOM 4188 OW WAT W 217 −18.371 5.558 23.186 1.00 61.33 O ATOM 4189 OW WAT W 218 −36.916 −12.555 18.483 1.00 34.96 O ATOM 4190 OW WAT W 219 −38.067 −30.490 11.033 1.00 53.39 O ATOM 4191 OW WAT W 220 −27.273 −1.669 22.879 1.00 60.24 O ATOM 4192 OW WAT W 221 −11.236 −0.446 20.423 1.00 57.47 O ATOM 4193 OW WAT W 222 −38.966 −27.165 28.768 1.00 48.50 O ATOM 4194 OW WAT W 223 −27.052 12.746 −2.875 1.00 54.30 O ATOM 4195 OW WAT W 224 −30.772 −4.221 20.144 1.00 59.60 O ATOM 4196 OW WAT W 225 −35.463 18.522 −0.106 1.00 60.87 O ATOM 4197 OW WAT W 226 −15.335 −26.347 9.375 1.00 44.12 O ATOM 4198 OW WAT W 227 2.572 −63.931 34.456 1.00 55.50 O ATOM 4199 OW WAT W 228 −16.786 −3.932 14.055 1.00 56.34 O ATOM 4200 OW WAT W 229 −9.872 −43.050 35.117 1.00 60.32 O ATOM 4201 OW WAT W 230 −37.011 −39.791 17.769 1.00 49.70 O ATOM 4202 OW WAT W 231 −22.797 24.445 17.168 1.00 56.75 O ATOM 4203 OW WAT W 232 0.236 −53.672 47.725 1.00 56.69 O ATOM 4204 OW WAT W 233 −26.383 1.765 2.046 1.00 49.47 O ATOM 4205 OW WAT W 234 −37.667 −27.913 10.243 1.00 48.69 O ATOM 4206 OW WAT W 235 −20.876 9.097 5.925 1.00 38.12 O ATOM 4207 OW WAT W 236 −15.798 −51.507 16.541 1.00 52.77 O ATOM 4208 OW WAT W 237 −32.462 −42.802 17.632 1.00 63.08 O ATOM 4209 OW WAT W 238 −41.086 16.561 32.871 1.00 51.80 O ATOM 4210 OW WAT W 239 −48.040 1.597 7.571 1.00 61.28 O ATOM 4211 OW WAT W 240 −36.279 11.284 −3.308 1.00 51.42 O ATOM 4212 OW WAT W 241 0.800 −54.597 50.511 1.00 56.31 O ATOM 4213 OW WAT W 242 −5.692 −65.042 32.265 1.00 50.05 O ATOM 4214 OW WAT W 243 −26.785 27.800 17.578 1.00 44.32 O ATOM 4215 OW WAT W 244 −32.984 −23.778 34.134 1.00 63.27 O ATOM 4216 OW WAT W 245 −4.702 −50.027 23.969 1.00 61.44 O ATOM 4217 OW WAT W 246 −20.411 −37.034 8.519 1.00 52.44 O ATOM 4218 OW WAT W 247 −28.495 −35.410 8.773 1.00 54.57 O ATOM 4219 OW WAT W 248 −29.060 14.857 34.292 1.00 62.48 O ATOM 4220 OW WAT W 249 −45.852 −2.251 25.140 1.00 52.91 O ATOM 4221 OW WAT W 250 −29.476 27.814 17.211 1.00 47.88 O ATOM 4222 OW WAT W 251 −13.400 11.724 9.810 1.00 67.92 O ATOM 4223 OW WAT W 252 −24.572 12.847 29.078 1.00 55.98 O ATOM 4224 OW WAT W 253 −33.478 −13.068 13.678 1.00 51.91 O ATOM 4225 OW WAT W 254 −21.816 −47.789 18.087 1.00 56.15 O ATOM 4226 OW WAT W 255 −24.270 −47.301 16.995 1.00 51.11 O ATOM 4227 OW WAT W 256 −43.497 −1.869 26.469 1.00 43.86 O ATOM 4228 OW WAT W 257 −29.905 −39.279 8.951 1.00 65.07 O ATOM 4229 OW WAT W 258 −44.193 7.821 11.648 1.00 54.88 O ATOM 4230 OW WAT W 259 −20.151 8.692 25.972 1.00 62.13 O ATOM 4231 OW WAT W 260 −7.665 −55.864 29.627 1.00 32.10 O ATOM 4232 OW WAT W 261 −5.906 −54.231 28.345 1.00 36.88 O ATOM 4233 OW WAT W 262 4.919 −62.040 41.051 1.00 36.45 O ATOM 4234 OW WAT W 263 −0.849 −56.077 31.823 1.00 33.23 O ATOM 4235 OW WAT W 264 −19.126 2.342 10.361 1.00 47.44 O ATOM 4236 OW WAT W 265 −20.601 −0.660 9.523 1.00 55.13 O ATOM 4237 OW WAT W 266 −25.961 1.094 7.475 1.00 51.67 O ATOM 4238 OW WAT W 267 −42.861 −22.137 19.876 1.00 43.74 O ATOM 4239 OW WAT W 268 −27.314 −12.468 20.423 1.00 42.25 O ATOM 4240 OW WAT W 269 −31.904 −11.852 21.052 1.00 36.97 O ATOM 4241 OW WAT W 270 −29.659 −11.156 19.733 1.00 42.01 O ATOM 4242 OW WAT W 271 −30.753 −11.559 16.905 1.00 44.60 O ATOM 4243 OW WAT W 272 −44.416 −3.925 6.661 1.00 56.55 O ATOM 4244 OW WAT W 273 −23.508 1.254 8.908 1.00 38.10 O ATOM 4245 OW WAT W 274 −30.753 −6.646 21.311 1.00 49.58 O ATOM 4246 OW WAT W 275 −30.261 −7.376 17.204 1.00 53.52 O ATOM 4247 OW WAT W 276 −32.174 −7.535 24.895 1.00 48.16 O ATOM 4248 OW WAT W 277 −43.043 8.906 19.447 1.00 43.35 O ATOM 4249 OW WAT W 278 −37.512 4.508 27.820 1.00 61.81 O ATOM 4250 OW WAT W 279 −36.941 0.000 0.000 0.50 55.93 O ATOM 4251 OW WAT W 280 −23.733 −24.004 33.240 1.00 62.39 O ATOM 4252 OW WAT W 281 −8.167 −30.315 8.553 1.00 68.84 O ATOM 4253 OW WAT W 282 −28.958 1.148 1.356 1.00 55.08 O ATOM 4254 OW WAT W 283 12.591 −61.771 32.569 1.00 46.20 O ATOM 4255 OW WAT W 284 −1.581 −49.613 41.371 1.00 58.06 O ATOM 4256 OW WAT W 285 −26.246 −48.282 18.626 1.00 59.66 O ATOM 4257 OW WAT W 286 2.540 −50.918 39.971 1.00 45.68 O ATOM 4258 OW WAT W 287 −3.856 −43.098 21.755 1.00 61.68 O ATOM 4259 OW WAT W 288 −36.642 23.118 18.736 1.00 62.84 O 

1. (canceled)
 2. (canceled)
 3. An antibody or antigen binding fragment thereof that binds to human CD27, wherein the antibody or antigen binding fragment comprises: a) a heavy chain variable region CDR1 comprising the amino acid sequence of SEQ ID NO: 1, wherein X₁=M; or an amino acid sequence differing from said sequence by 1, 2, or 3 conservative substitutions; b) a heavy chain variable region CDR2 comprising the amino acid sequence of SEQ ID NO: 2, wherein X₁=N, X₂=T, X₃=N and X₄=T, or an amino acid sequence differing from said sequence by 1, 2, or 3 conservative substitutions; c) a heavy chain variable region CDR3 comprising the amino acid sequence of SEQ ID NO: 3, wherein X₁=M, or an amino acid sequence differing from said sequence by 1, 2, or 3 conservative substitutions; d) a light chain variable region CDR1 comprising the amino acid sequence of SEQ ID NO: 4, wherein X₁=M, or an amino acid sequence differing from said sequence by 1, 2, or 3 conservative substitutions; e) a light chain variable region CDR2 comprising the amino acid sequence of SEQ ID NO: 5, wherein X₁=D and X₂=T, or an amino acid sequence differing from said sequence by 1, 2, or 3 conservative substitutions; and f) a light chain variable region CDR3 comprising the amino acid sequence of SEQ ID NO: 6, wherein X₁=W, X₂=N and X₃=S, or an amino acid sequence differing from said sequence by 1, 2, or 3 conservative substitutions.
 4. An antibody or antigen binding fragment thereof that binds to human CD27 comprising a light chain immunoglobulin variable region, a heavy chain immunoglobulin variable region or both a light chain and a heavy chain immunoglobulin variable region selected from the group consisting of: a) an antibody or antigen binding fragment thereof comprising a variable heavy chain comprising the amino acid sequence of SEQ ID NO:7 or an amino acid sequence differing from SEQ ID NO: 7 by 1, 2, or 3 conservative substitutions and/or a variable light chain comprising the amino acid sequence of SEQ ID NO: 8 or an amino acid sequence differing from SEQ ID NO: 8 by 1, 2, or 3 conservative substitutions; b) an antibody or antigen binding fragment thereof comprising a variable heavy chain comprising the amino acid sequence of SEQ ID NO: 9 or an amino acid sequence differing from SEQ ID NO: 9 by 1, 2, or 3 conservative substitutions and/or a variable light chain comprising the amino acid sequence of SEQ ID NO: 14 or an amino acid sequence differing from SEQ ID NO: 14 by 1, 2, or 3 conservative substitutions; c) an antibody or antigen binding fragment thereof comprising a variable heavy chain comprising the amino acid sequence of SEQ ID NO: 32 or an amino acid sequence differing from SEQ ID NO: 32 by 1, 2, or 3 conservative substitutions and/or a variable light chain comprising the amino acid sequence of SEQ ID NO: 33 or an amino acid sequence differing from SEQ ID NO: 33 by 1, 2, or 3 conservative substitutions; d) an antibody or antigen binding fragment thereof comprising a variable heavy chain comprising the amino acid sequence of SEQ ID NO: 34 or an amino acid sequence differing from SEQ ID NO: 34 by 1, 2, or 3 conservative substitutions and/or a variable light chain comprising the amino acid sequence of SEQ ID NO: 35 or an amino acid sequence differing from SEQ ID NO: 35 by 1, 2, or 3 conservative substitutions; e) an antibody or antigen binding fragment thereof comprising a variable heavy chain comprising the amino acid sequence of SEQ ID NO: 39 or an amino acid sequence differing from SEQ ID NO: 39 by 1, 2, or 3 conservative substitutions and/or a variable light chain comprising the amino acid sequence of SEQ ID NO: 40 or an amino acid sequence differing from SEQ ID NO: 40 by 1, 2, or 3 conservative substitutions; f) an antibody or antigen binding fragment thereof comprising a variable heavy chain selected from the group consisting of SEQ ID Nos: 10-13 or an amino acid sequence differing from one of SEQ ID Nos: 10-13 by 1, 2, or 3 conservative substitutions and/or a variable light chain selected from the group consisting of any one of SEQ ID Nos: 15-18 or an amino acid sequence differing from one of SEQ ID Nos: 15-18 by 1, 2, or 3 conservative substitutions; g) an antibody or antigen binding fragment thereof comprising a variable heavy chain comprising the amino acid sequence of SEQ ID NO: 10 or an amino acid sequence differing from SEQ ID NO: 10 by 1, 2, or 3 conservative substitutions and/or a variable light chain comprising the amino acid sequence of SEQ ID NO: 15 or an amino acid sequence differing from SEQ ID NO: 15 by 1, 2, or 3 conservative substitutions; h) an antibody or antigen binding fragment thereof comprising a variable heavy chain comprising at least 90% identity to any one of SEQ ID Nos: 10-13 and/or a variable light chain comprising at least 90% identity to any one of SEQ ID Nos: 15-18; i) an antibody or antigen binding fragment thereof comprising a variable heavy chain comprising at least 90% identity to any one of SEQ ID Nos: 10-13 and/or a variable light chain comprising at least 90% identity to any one of SEQ ID Nos: 15-18, wherein any sequence variations occur in the framework regions of the antibody or antigen binding fragment; j) an antibody or antigen binding fragment thereof comprising a variable heavy chain comprising 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions with respect to any one of SEQ ID Nos: 10-13 and/or a variable light chain comprising 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions with respect to any one of SEQ ID Nos: 15-18; and k) an antibody or antigen binding fragment thereof comprising a variable heavy chain comprising 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions with respect to any one of SEQ ID Nos: 10-13 and/or a variable light chain comprising 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions with respect to any one of SEQ ID Nos: 15-18, wherein the amino acid substitutions occur in the framework regions of the antibody or antigen binding fragment.
 5. The antibody or antigen binding fragment thereof of claim 4, comprising a light chain and a heavy chain immunoglobulin variable region selected from the group consisting of: a) a variable heavy chain comprising the amino acid sequence of SEQ ID NO: 9 and a variable light chain comprising the amino acid sequence of SEQ ID NO: 14; b) a variable heavy chain comprising the amino acid sequence of SEQ ID NO: 32 and a variable light chain comprising the amino acid sequence of SEQ ID NO: 33; and c) a variable heavy chain comprising the amino acid sequence of SEQ ID NO: 34 and a variable light chain comprising the amino acid sequence of SEQ ID NO:
 35. 6. (canceled)
 7. (canceled)
 8. (canceled)
 9. (canceled)
 10. (canceled)
 11. (canceled)
 12. (canceled)
 13. An antibody or antigen binding fragment thereof that binds the same epitope of human CD27 as an antibody comprising the variable heavy chain of SEQ ID NO: 10 and the variable light chain of SEQ ID NO: 15; or binds the same epitope of human CD27 as an antibody comprising the variable heavy chain of SEQ ID NO:7 and the variable light chain of SEQ ID NO:8.
 14. An antibody or antigen binding fragment thereof, wherein, when bound to human CD27, binds at least one residue selected from the group consisting of Leu18, Asp34, Gln35, and Lys38 of SEQ ID NO:
 19. 15. The antibody or antigen binding fragment thereof of claim 14, wherein, when bound to human CD27, binds at least one residue selected from the group consisting of Gln35 and Lys38 of SEQ ID NO:
 19. 16. The antibody or antigen binding fragment of claim 14, wherein the antibody or antigen binding fragment thereof further binds one or more residues selected from the group consisting of Pro8, Glu9, His11, Lys17, His36, and Arg37 of SEQ ID NO:
 19. 17. The antibody or antigen binding fragment of claim 14, wherein the antibody or antigen binding fragment thereof further binds one or more residues selected from the group consisting of Glu9, Lys17, His36, and Arg37 of SEQ ID NO:
 19. 18. The antibody or antigen binding fragment thereof of claim 14, wherein the antibody or antigen binding fragment thereof binds residues Pro8, Glu9, His11, Lys17, Leu18, Asp34, Gln35, His36, Arg37 and Lys38 of SEQ ID NO:
 19. 19. The antibody or antigen binding fragment thereof of claim 14, wherein the antibody or antigen binding fragment thereof binds residues Glu9, Lys17, Leu18, Asp34, Gln35, His36, Arg37 and Lys38 of SEQ ID NO:
 19. 20. (canceled)
 21. The antibody or antigen binding fragment of claim 3, which is a humanized antibody comprising two heavy chains and two light chains, and is an IgG isotype.
 22. The antibody or antigen binding fragment of claim 3, which is an antibody of the IgG1 isotype.
 23. The antibody or antigen binding fragment of claim 3, which is an antibody of the IgG4 isotype.
 24. The antibody or antigen binding fragment of claim 3, wherein the antibody comprises the heavy chain constant domain of SEQ ID NO:
 30. 25. The antibody or antigen binding fragment of claim 3, wherein the antibody comprises the heavy chain constant domain of SEQ ID NO:
 28. 26. (canceled)
 27. The antibody or antigen binding fragment of claim 3, wherein the antibody or antigen binding fragment thereof comprises a glycosylation pattern characteristic of expression by a mammalian cell.
 28. The antibody or antigen binding fragment of claim 3, wherein the antibody or antigen binding fragment thereof comprises a glycosylation pattern characteristic of expression by a CHO cell.
 29. (canceled)
 30. (canceled)
 31. (canceled)
 32. (canceled)
 33. (canceled)
 34. (canceled)
 35. (canceled)
 36. (canceled)
 37. (canceled)
 38. (canceled)
 39. (canceled)
 40. (canceled)
 41. (canceled)
 42. (canceled)
 43. (canceled)
 44. (canceled)
 45. (canceled) 